RU2776369C1 - Combinations for treating nash/nafld and associated diseases - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: group of inventions relates to the field of medicine and pharmaceuticals and can be used for treating a disease or condition selected from fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with liver cirrhosis, and non-alcoholic steatohepatitis with liver cirrhosis and with hepatocellular carcinoma or with a metabolic disease. Proposed is a pharmaceutical composition for treating the above diseases, including (S)-2-(5-((3-ethoxypyridine-2-yl)oxy)pyridine-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide or a pharmacologically acceptable salt thereof in combination at least with 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridine-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, each compound in a therapeutically effective amount, mixed with a pharmacologically acceptable excipient. The group of inventions also relates to methods for treating said diseases, including the administration of a therapeutically effective amount of the above compounds.

EFFECT: significant reduction in the level of triglycerides in plasma and liver.

25 cl, 24 dwg, 8 tbl, 2 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The invention relates to new pharmaceutical compositions, including (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide or a pharmaceutically acceptable salt thereof and, in the same or separate composition, 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1 '-carbonyl)-6-methoxypyridin-2-yl)benzoic acid, or a pharmaceutically acceptable salt thereof, for the treatment of non-alcoholic fatty liver disease (NAFLD) and related diseases such as non-alcoholic steatohepatitis (NASH) and metabolic diseases.

BACKGROUND OF THE INVENTION

Non-alcoholic steatohepatitis (NASH) is the clinical and histological subtype of non-alcoholic fatty liver disease (NAFLD defined as >5% fatty liver disease) that is associated with increased mortality from various causes, cirrhosis of the liver and end-stage liver disease, increased mortality due to cardiac -vascular complications, and an increase in the incidence of oncological complications, both related and non-liver related (Sanyal et al., Hepatology 2015;61(4):1392-1405). NAFLD is the hepatic manifestation of the metabolic syndrome and is a spectrum of liver conditions including steatosis (fatty liver), NASH, fibrosis, cirrhosis, and ultimately hepatocellular carcinoma. NAFLD and NASH are considered to be the main fatty liver diseases, since among patients with these diseases, the largest proportion are patients with elevated liver lipids. The severity of NAFLD/NASH determines the presence of lipids, inflammatory cell infiltrate, ballooning of liver cells, and the extent of fibrosis. Currently, treatment options are limited to the treatment of comorbid conditions (EASL-EASD-EASO Clinical Practice Guidelines, J. Hepatol. 2016;64(6):1388-1402).

Changes in lipid metabolism are thought to contribute to the molecular pathogenesis of NAFLD and NASH. Steatosis is a necessary but not sufficient component of the pathogenesis of NASH (Day C., and James O., Hepatology. 1998; 27(6):1463-6). Accordingly, numerous studies have shown that the severity of steatosis determines the risk of concomitant steatohepatitis, as well as the risk of developing liver cirrhosis (Sorensen et al., Lancet. 1984; 2(8397): 241-4; Wanless I. and Lentz J., Hepatology 1990; 12(5): 1106-10; Reeves H., et al J. Hepatol 1996; 25(5): 677-83). Hepatic steatosis is a consequence of an imbalance between the production/absorption of TG by the liver and their clearance/excretion from the liver (Cohen J.C., et al., Science. 2011; 332(6037):1519-1523). It is expected that the reduction of steatosis, the metabolic factor underlying the development of NAFLD/NASH, will lead to subsequent improvements in liver inflammation and fibrosis.

Acetyl-CoA carboxylase (ACC) and diacylglycerol acyltransferase-2 (DGAT2) are two key enzymes that regulate lipid metabolism. AKK in the process of primary lipogenesis (de novo lipogenesis - DNL) catalyze its vital and rate-limiting stage (Saggerson D., Annu. Rev. Nutr. 2008; 28:253-72.). In addition, ACC also regulates mitochondrial beta-oxidation of fatty acids through allosteric regulation of the enzyme carnitine palmitoyltransferase 1 (CPT1) (Saggerson, 2008; Waite M, and Wakil S.J. J. Biol. Chem. 1962;237:2750-2757 .). The findings also suggest that suppression of DNL by inhibition of ACC may directly reduce inflammation by inhibiting the formation of inflammatory interleukin-17 (interleukin-17 - IL-17), which secretes T-cells of the T-helper line (Th17 cells), and promote the development of anti-inflammatory FoxP3(+) regulatory T cells (Treg) (Berod L., et al. Nat. Med. 2014; 20(11): 1327-33).

Inhibition of ACC activity is thought to be beneficial to NASH patients through at least two independent mechanisms. As noted above, there is a marked increase in hepatic DNL in patients with NAFLD, and normalization of this increased secretion through pharmacological inhibition of hepatic ACC is expected to reduce steatosis. In addition, the action of AKK inhibitors, which cause an increase in fatty acid oxidation, may also contribute to the reduction of liver fat. Accordingly, AKK inhibitors have been shown to inhibit DNL. See Griffith D.A., et al. J. Med. Chem. 2014;57(24):10512-10526; Kim C.W., et al. Cell Metab. 2017;26, 394-406; Stiede K., et al. hepatology. 2017;66(2):324-334; Lawitz, E.J., et al. Clin Gastroenterol Hepatol. 2018 (https://doi.org/10.1016/j.cgh.2018.04.042). In addition, inhibition of DNL in T-secreting IL-17 is expected to suppress liver inflammation by limiting the production of inflammatory Th17 cells (Berod et al., 2014), a metabolic pathway that may be important in the pathogenesis of NASH (Rau M., et al. J. Immunol. 2016;196(1):97-105) and is favorable for the development of anti-inflammatory Treg cells. In addition, ACC inhibition may reduce stellate cell activation and fibrosis (Ross et al., 2019).

Triglycerides or triacylglycerols (TG) are the main form of energy storage (storage) in mammals. TGs are formed through the sequential esterification of glycerol with three fatty acids of different chain lengths and degrees of saturation (Coleman, R. A., and Mashek, D. G. 2011. Chem. Rev. 111: 6359-6386). TG synthesized in the intestine or liver are assembled into chylomicrons or very low-density lipoprotein (VLDL), respectively, and exported to peripheral tissues, where they are hydrolyzed by lipoprotein lipase (LPL) to their constituent fatty acids and glycerin. The resulting non-esterified fatty acids (NEFA) can be further metabolized for energy or re-esterified and stored.

Under normal physiological conditions, energetically dense TG remains sequestered in various fat depots until the need for its release arises, after which it is hydrolyzed to glycerol and free fatty acids, which are then released into the bloodstream. This process is tightly regulated by the opposing actions of insulin and hormones such as catheolamines, which promote the deposition and mobilization of TG stores under various physiological conditions. After a meal, insulin inhibits lipolysis, thus inhibiting the release of energy in the form of NEFA and ensuring proper storage of dietary lipids in fat depots. However, in patients with type 2 diabetes, the ability of insulin to suppress lipolysis is increased and the flux of NEFA from adipocytes is increased inappropriately. This in turn leads to increased lipid delivery to tissues such as muscle and liver. In the absence of energy demand, TG and other lipid metabolites, such as diacylglycerol (DAG), can accumulate and cause loss of insulin sensitivity (Erion, D.M., and Shulman, G.I. 2010. Nat Med 16: 400-402). Insulin resistance in muscle is characterized by reduced glucose uptake and glycogen storage, while in the liver, loss of insulin signaling pathways leads to dysregulation of glucose production and overproduction of TG-rich VLDLs, a hallmark of type 2 diabetes (Choi, S.H., and Ginsberg , H. N. 2011 Trends Endocrinol Metab 22: 353-363). Increased secretion of TG-rich VLDL, the so-called VLDL1 particles, is thought to stimulate the production of small dense low-density lipoprotein (sdLDL), a pro-atherogenic LDL subfraction that is associated with an increased risk of coronary heart disease (St-Pierre , A. C. et al 2005 Arterioscler Thromb Vasc Biol 25: 553-559).

In mammals, two diacylglycerol acyltransferase (DGAT) enzymes (DGAT1 and DGAT2) have been characterized. Although these enzymes catalyze the same enzymatic reaction, their respective amino acid sequences are not related and belong to different gene families. Mice with a disruption of the gene encoding DGAT1 are resistant to diet-induced obesity and exhibit increased energy intake and increased activity (Smith, S. J. et. al., 2000. Nat Genet 25: 87-90). Dgat1 −/− mice exhibit unregulated postaborpative chylomicron release and lipid accumulation in enterocytes (Buhman, K.K. et.al. 2002. J. Biol. Chem. 277: 25474-25479). It is suggested that the metabolically favorable phenotype observed in these mice is due to the loss of DGAT1 expression in the gut (Lee, B., et.al. 2010. J. Lipid Res. 51: 1770-1780). It is important to note that despite impaired lactation in female Dgat1 -/- mice, these animals retain the ability to synthesize TG, indicating the existence of additional DGAT enzymes. This observation and isolation of a second DGAT from the fungus Mortierella rammaniana led to the identification and characterization of DGAT2 (Yen, C.L. et al. 2008. J. Lipid Res. 49: 2283-2301).

DGAT2 is highly expressed in the liver and adipose tissue and, unlike DGAT1, exhibits exceptional substrate specificity for DAG (Yen, C.L., 2008). Deletion of the DGAT2 gene in rodents leads to impaired intrauterine development, severe lipemia, impaired skin barrier function, and early postpartum death (Stone, S. J. et.al. 2004. J. Biol. Chem. 279: 11767-11776). Because of the lethality caused by the loss of DGAT2, much of the knowledge about the physiological role of DGAT2 comes from studies done with antisense oligonucleotides (ASO) in rodent models of metabolic disease. Under these conditions, inhibition of hepatic DGAT2 leads to an improvement in the plasma lipoprotein profile (decrease in total cholesterol and TG) and in a decrease in liver lipid load, which is accompanied by improved insulin sensitivity and glucose control in the body (Liu, Y. et.al. 2008. Biochim Biophys Acta 1781: 97-104; Choi, C. S. et al. 2007. J. Biol. Chem. 282: 22678-22688; Yu, X. X. et al. 2005. Hepatology 42: 362-371). Although the molecular mechanisms underlying these observations are not fully understood, it is clear that downregulation of DGAT2 leads to downregulation of several genes encoding proteins involved in lipogenesis, including sterol-regulating element binding proteins 1c (SREBP1c) and stearoyl-CoA desaturase 1 (stearoyl CoA-desaturase 1 - SCD1) (Choi, 2007; Yu, 2005). In parallel, oxidative metabolic pathways are induced, as evidenced by the increased expression of genes such as carnitine palmitoyl transferase 1 (CPT1) (Choi, 2007). The net result of these changes is a decrease in hepatic DAG and TG lipid levels, which in turn leads to an improvement in hepatic insulin sensitivity. In addition, inhibition of DGAT2 suppresses hepatic secretion of c VLDL TG and lowers circulating cholesterol levels. Finally, the levels of apolipoprotein B (apolipoprotein B - APOB) were reduced, possibly due to a decrease in the supply of TG for lipidation of the newly synthesized APOB protein (Liu, 2008; Yu, 2005). The beneficial effects of DGAT2 inhibition on both glycemic control and plasma cholesterol profile suggest that this target could be used in the treatment of metabolic disease (Choi, 2007). In addition, the fact that inhibition of DGAT2 activity leads to a decrease in hepatic lipid accumulation suggests that inhibitors of this enzyme may be useful in the treatment of NASH.

In view of the above, there is a need for drugs, for example drugs for oral administration, containing the combination of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-( tetrahydrofuran-3-yl)pyrimidine-5-carboxamide (DGAT2 inhibitor) or a pharmaceutically acceptable salt thereof, and in the same or separate formulation 4-(4-(1-isopropyl-7-oxo-1,4,6,7- tetrahydrospiro[indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid (AKKI inhibitor) or a pharmaceutically acceptable salt thereof. The specific combinations described herein meet the present need.

SUMMARY OF THE INVENTION

The present invention relates to a pharmaceutical composition comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof in combination with at least 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl) -6-methoxypyridin-2-yl)-benzoic acid or a pharmaceutically acceptable salt thereof, both in therapeutically effective amounts, in admixture with a pharmaceutically acceptable excipient.

The present invention also relates to a pharmaceutical composition comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5- carboxamide or a pharmaceutically acceptable salt thereof and 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridine -2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable excipient.

The present invention also relates to a method of treating a disease or condition selected from fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with liver cirrhosis, and non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma or metabolic disease. wherein said method comprises administering to a patient in need of such treatment a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3 -yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof, in combination with a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'- piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof.

The present invention also relates to a method for reducing at least one point in scoring systems for the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, reducing the level of serum markers of activity of non-alcoholic steatohepatitis, reducing the activity of the disease of non-alcoholic steatohepatitis, or reducing the medical consequences of non-alcoholic steatohepatitis in humans, comprising the stage administering to a patient in need of such a reduction a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin- 5-carboxamide, or a pharmaceutically acceptable salt thereof, in combination with at least a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine] -1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof.

The present invention also relates to a method of treating fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver, or non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma in humans, comprising the step of administering to a person in need of such treatment, a therapeutically effective amount of the first and second compositions, and optionally a third composition, wherein

i. the first composition comprises (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient;

ii. the second composition comprises 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl ) benzoic acid or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient; and

iii. the third composition comprises a pharmaceutical agent selected from the group consisting of an anti-inflammatory agent, an antidiabetic agent, an antifibrotic agent, an antisteatotic agent, a cholesterol/lipid modulating agent, and an antidiabetic agent, and a pharmaceutically acceptable excipient.

The present invention also relates to a method of treating fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver, or non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma in humans, comprising the step of administering to a person in need of such treatment, a therapeutically effective amount of two separate pharmaceutical compositions, including

i. a first composition which comprises (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide or a pharmaceutically an acceptable salt in a therapeutically effective amount in admixture with a pharmaceutically acceptable excipient;

ii. the second composition, which includes 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridine-2 -yl)benzoic acid or a pharmaceutically acceptable salt thereof in a therapeutically effective amount in admixture with a pharmaceutically acceptable excipient; and optionally

iii. a third composition comprising at least one additional pharmaceutical agent selected from the group consisting of a GLP-1R agonist, a KHK inhibitor, or an FXR agonist, and a pharmaceutically acceptable excipient.

The invention also relates to a method for the treatment of heart failure, congestive heart failure, coronary heart disease, peripheral vascular disease, renovascular disease, pulmonary hypertension, vasculitis, acute coronary syndromes and cardiovascular risk modification, comprising administering to a person in need of such treatment, therapeutically effective amounts of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof in combination with at least a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridine -2-yl)benzoic acid or a pharmaceutically acceptable salt thereof.

The invention also relates to a method for the treatment of obesity, type I diabetes, type II diabetes mellitus, idiopathic type I diabetes (type Ib), latent autoimmune diabetes in adults (latent autoimmune diabetes in adults - LADA), juvenile diabetes (early-onset diabetes - EOD ) Type 2, youth-onset atypical diabetes (YOAD), adult type juvenile diabetes (maturity onset diabetes of the young - MODY), diabetes associated with malnutrition, gestational diabetes, coronary heart disease, ischemic stroke, restenosis after angioplasty, peripheral vascular disease, intermittent claudication, myocardial infarction, dyslipidemia, postprandial lipemia, conditions of impaired glucose tolerance (IGT), conditions of impaired fasting plasma glucose, metabolic acidosis, ketosis, arthritis, diabetic retinopathy , macular degeneration, cataracts, diabetic nephropathy ii, glomerulosclerosis, chronic renal failure, diabetic neuropathy, metabolic syndrome, syndrome X, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, insulin resistance, impaired glucose metabolism, skin and connective tissue disorders, foot ulcers and ulcerative colitis, endothelial dysfunction and vascular elasticity disorders, hyperapolipoproteinemia B and maple syrup odor urinary disease comprising administering to a person in need of such treatment a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N- (tetrahydrofuran-3-yl)pyrimidine-5-carboxamide, or a pharmaceutically acceptable salt thereof, in combination with at least a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[ indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)-benzoic acid or a pharmaceutically acceptable salt thereof.

The invention also relates to a method for the treatment of hepatocellular carcinoma, clear cell kidney cancer, head and neck squamous cell carcinoma, colorectal adenocarcinoma, mesothelioma, gastric adenocarcinoma, adrenocortical carcinoma, papillary cell kidney cancer, cervical and endocervical carcinoma, urothelial carcinoma of the bladder, adenocarcinoma of the lung comprising administering to a person in need of such treatment a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl) pyrimidine-5-carboxamide, or a pharmaceutically acceptable salt thereof, in combination with at least a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'- piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof.

It should be understood that the foregoing general description and the following detailed description are illustrative and explanatory only and do not limit the claimed invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a characteristic X-ray powder diffraction pattern of the crystalline form 1 of the DGAT2i compound of Example 1 (vertical axis: intensity (number of pulses per second); horizontal axis: two theta (degrees)).

Figure 2 is a characteristic X-ray powder diffraction pattern of the crystalline form 2 of the DGAT2i compound of Example 1 (vertical axis: intensity (number of pulses per second); horizontal axis: two theta (degrees)).

Figure 3 is an illustrative X-ray powder diffraction pattern (XRD) of Compound A Form 1 obtained on a Bruker AXS D4 Endeavor diffractometer equipped with a Cu radiation source.

Figure 4 is an exemplary Raman spectrum (Raman spectrum) of Compound A Form 1 obtained using a Nicolet NXR FT-Raman accessory attached to an FT-IR bench.

Figure 5 is an exemplary ¹³ C NMR solid state ( ¹³ C ssNMR) spectrum of Compound A Form 1 obtained using a Bruker-BioSpin CPMAS probe placed in a Bruker-BioSpin Avance III 500 MHz NMR spectrometer ( ¹ H frequency).

Figure 6 is an exemplary XRD of form 2 of compound A obtained on a Bruker AXS D4 Endeavor diffractometer equipped with a copper source.

Figure 7 is an exemplary Compound A Form 2 Raman spectrum obtained using a Nicolet NXR FT-Raman accessory attached to an FT-IR bench.

Figure 8 is an exemplary ¹³ C ssNMR spectrum of form 2 of compound A obtained on a Bruker-BioSpin CPMAS probe located in a Bruker-BioSpin Avance III 500 MHz NMR spectrometer ( ¹ H frequency).

Figure 9 shows an exemplary single crystal structure of form 2 of compound A.

Figure 10 shows the effect of oral administration of Compound A and Compound D alone and in combination on postprandial plasma triglyceride levels in Sprague Dawley rats fed a Western diet.

Figure 11 shows the effect of oral administration of Compound A and Compound D alone and in combination on fasting plasma triglyceride levels in Sprague Dawley rats fed a Western diet.

Figure 12 shows the effect of administration of Compound A and Compound D, alone and in combination, on the nuclear localization of SREBP-1 in Western diet-fed rats.

Figure 13 shows the effect of administering Compound A and Compound D alone and in combination on the expression of lipogenic genes, in particular acetyl-CoA carboxylase (AKK1), in the liver of Western-fed rats.

Figure 14 shows the effect of administering Compound A and Compound D alone and in combination on the expression of lipogenic genes, in particular fatty acid synthase (FASN), in the liver of Western-fed rats.

Figure 15 shows the effect of administering Compound A and Compound D alone and in combination on the expression of lipogenic genes, in particular stearoyl-CoA desaturase (SCD1), in the liver of Western diet-fed rats.

Figure 16 shows the effect of administering Compound A and Compound D alone and in combination on the expression of lipogenic genes, in particular sterol-regulating element binding protein 1c (SREBP-1c), in the liver of rats fed a Western diet.

The figure 17 shows the effect of the administration of compound A and compound D alone and in combination on the expression of lipogenic genes, in particular proprotein convertase subtilisin/kexin type 9 (PCSK9), in the liver of rats treated with food according to the western diet.

18 shows the effect of oral administration of Compound A and Compound D alone and in combination on liver triglyceride levels in Sprague Dawley rats fed a Western diet.

Figure 19 shows the effect of oral administration of Compound A and Compound D, alone and in combination, on liver elasticity, a marker of inflammation and liver fibrosis, in male Wistar-Hann rats fed a low-choline, high-fat diet. (choline deficient and high fat diet - CDAHFD).

Figure 20 shows the effect of oral administration of Compound A and Compound D alone and in combination on the immunohistochemistry of alpha smooth muscle actin (αSMA), a marker of myofibroblast activation and fibrogenesis, in the liver of male Wistar Hann rats fed a accordance with CDAHFD.

Figure 21 shows the effect of oral administration of Compound A and Compound D alone and in combination on Picrosirius red staining in the liver of CDAHFD-fed male Wistar Hann rats.

Figure 22 shows the effect of oral administration of Compound A and Compound D alone and in combination on alpha-smooth muscle actin (αSMA) gene expression in the liver of CDAHFD-fed male Wistar Hann rats.

Figure 23 shows the effect of oral administration of Compound A and Compound D alone and in combination on collagen 1A1 gene expression in the liver of male Wistar Hann rats fed according to CDAHFD.

Figure 24 shows the effect of oral administration of Compound A and Compound D alone and in combination on the staining of ionized calcium binding adapter molecule 1 in CDAHFD-fed male Wistar Hann rats.

DETAILED DESCRIPTION OF THE INVENTION

The invention can be more easily understood by referring to the following detailed description of typical examples of embodiments of the invention and examples included therein.

It should be borne in mind that the present invention is not limited to specific methods of synthesis, which, of course, may vary. It should also be borne in mind that the terminology used here is only intended to describe specific embodiments, and not to limit them. In this description and in the appended claims, a number of terms will be mentioned, the meaning of which is defined below.

The use in the present description of the singular means the plural. The term "other" when used in the present description, may mean at least the second or greater in a row.

The term "about" is a relative term denoting an approximation within plus or minus 10% of the nominal value, which means in one embodiment up to plus or minus 5%, in another embodiment up to plus or minus 2%. For the scope of the present invention, this level of approximation is appropriate, unless specifically stated that the value requires a narrower range of values.

The term "compounds" as used herein includes any pharmaceutically acceptable derivative or variant, including conformational isomers (e.g., cis and trans isomers) and all optical isomers (e.g., enantiomers and diastereomers), racemic, diastereomeric, and other mixtures. such isomers, as well as solvates, hydrates, isomorphs, polymorphs, tautomers, esters, salt forms and prodrugs. The term "prodrug" refers to compounds that are drug precursors and, upon administration, release the drug in vivo by some chemical or physiological process (e.g., the prodrug is converted to the desired dosage form at physiological pH or by the action of enzymes). Typical examples of prodrugs upon cleavage release the corresponding free acid, and such hydrolysable ester-forming residues of the compounds of the present invention include, but are not limited to, a carboxyl moiety in which the free hydrogen is replaced by (C ₁ -C ₄ )alkyl, (C ₂ -C ₇ )alkanoyloxymethyl, 1-(alkanoyloxy)ethyl containing from 4 to 9 carbon atoms, 1-methyl-1-(alkanoyloxy)ethyl containing from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl containing from 3 to 6 carbon atoms , 1-(alkoxycarbonyloxy)ethyl containing from 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl containing from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl containing from 3 to 9 carbon atoms, 1-(N-(alkoxycarbonyl(amino)ethyl) containing from 4 to 10 carbon atoms, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl, di-N, N-(C ₁ -C ₂ )alkylamino (C ₂ -C ₃ )alkyl (such as β-dimethylaminoethyl), carbamoyl-(C ₁ -C ₂ )alkyl, N, N-di(C ₁ -C ₂ )alk ylcarbamoyl-(C ₁ -C ₂ )alkyl; and piperidino, pyrrolidino or morpholino(C ₂ -C ₃ )alkyl.

» или волнистая линия «

», когда используются в настоящем описании, означают точку присоединения заместителя к другой группе.Arrow pointer «

"or wavy line"

", when used in the present description, mean the point of attachment of the substituent to another group.

The term "patient" refers to warm-blooded animals such as, for example, guinea pigs, mice, rats, gerbils, cats, rabbits, dogs, cattle, goats, sheep, horses, monkeys, chimpanzees, and humans. The term "mammal" means "patient".

The term "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients in the formulation and/or with the mammal to be treated with them.

Terms relating to the administration of pharmaceuticals, when used in the present description, have the following meanings: "QD" means once a day, "BID" means twice a day.

The terms "reaction-inert solvent" and "inert solvent" as used herein refer to a solvent or mixture of solvents that does not react with the starting materials, reagents, intermediates or reaction products and does not adversely affect the yield of the target product.

The term "selectivity" or "selective" as used herein refers to a greater effect of a compound in a first assay compared to that of the same compound in a second assay. For example, for "gut selective" compounds, the first assay is for the gut half-life of the compound and the second assay is for the liver half-life of the compound.

The term "therapeutically effective amount" means an amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6- methoxypyridin-2-yl)benzoic acid (compound A) in combination with an amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3 -yl)pyrimidine-5-carboxamide (compound D), and optionally in combination with an amount of other compound(s) that treats the particular disease, condition or disorder described in the present invention.

4-(4-(1-Isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid is a selective ACC inhibitor and was prepared as the free acid in Example 9 of US Patent No. 8,859,577, which is US National Publication of International Patent Application No. PCT/IB2011/054119, and the contents of both of these documents are incorporated herein by reference in their entirety. for all purposes. The crystalline forms of the compound are described in International Patent Application No. PCT/IB2018/058966 published as WO 2019/102311 on May 31, 2019.

(S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide is a DGAT2 inhibitor and is described as a compound Example 1 in US Patent Application Publication No. 2018-0051012A1, the contents of which are incorporated herein by reference for all purposes.

The term "treatment", "treat" or "treatment", when used in the present description, means preventive, i.e. prophylactic, treatment, palliative treatment, i.e. attenuating, alleviating or slowing down the development in a patient of a disease (or condition) or any tissue damage, associated with a disease (or condition); and reversal of development, when not only the disease (or condition) of the patient is alleviated, but the condition of tissues damaged in connection with the disease (or condition) of the patient improves, compared with their condition at the beginning of treatment. This latter may occur when there is, but is not limited to, one or more of the following: demonstrating resolution of NASH and/or improvement in fibrosis scores based on liver biopsy; reduction in disease progression to cirrhosis, hepatocellular carcinoma, and/or other liver-related endpoints; decrease or improvement in the level of serum or imaging markers of non-alcoholic steatohepatitis activity; reduction or improvement in the activity of non-alcoholic steatohepatitis; or reducing the medical consequences of non-alcoholic steatohepatitis.

It appears that the administration of an AKC inhibitor may have an effect beneficial in reducing hepatic TG and possibly other beneficial effects in the treatment of NASH. Elevated circulating TG levels have been reported to be a mechanistic consequence of hepatic ACC inhibition (Kim et al., 2017), although doses of ACC inhibitors that only partially inhibit DNL may not increase circulating TG levels (Bergman et al., (2018) J. of Hepatology, Volume 68, S582). WO2016/112305 describes methods of treating, stabilizing or reducing the severity or development of non-alcoholic fatty liver disease using an ACC inhibitor alone or with one or more additional therapeutic agents.

It was found that the introduction of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridine-2 -yl)benzoic acid, optionally in the form of a pharmaceutically acceptable salt, can lead to increased blood levels of TG (usually measured in plasma) in Sprague Dawley rats fed a Western diet, as observed in human patients.

The compounds of the present invention may contain asymmetric or chiral centers and therefore exist in various stereoisomeric forms. Unless otherwise indicated, all stereoisomeric forms of the compounds of the present invention, as well as mixtures thereof, including racemic mixtures, are intended to form part of the invention. In addition, the invention covers all geometric and positional isomers. For example, if a compound of the present invention includes a double bond or a condensed ring, cis- and transforms, as well as mixtures thereof, are included within the scope of the present invention.

The chiral compounds of the present invention (and their chiral precursors) can be prepared in an enantiomerically enriched form using chromatography, typically high pressure liquid chromatography (HPLC) or supercritical fluid chromatography (SLC), on a resin with an asymmetric stationary phase and with a mobile phase, containing a hydrocarbon, typically heptane or hexane, containing 0 to 50% isopropanol, typically 2 to 20%, and 0 to 5% alkylamine, typically 0.1% diethylamine (DEA) or isopropylamine. An enantiomerically enriched mixture is obtained by concentrating the eluent.

Mixtures of diastereomers can be separated into individual diastereoisomers using differences in their physicochemical properties by methods well known to those skilled in the art, such as chromatography and/or fractional crystallization. Enantiomers can be separated by converting an enantiomeric mixture to a diastereomeric mixture by reacting with a suitable optically active compound (e.g., a chiral auxiliary such as a chiral alcohol or Mosher acid chloride), separating the diastereoisomers, and converting (e.g., hydrolyzing) the separated diastereoisomers to the corresponding pure enantiomers. Enantiomers can also be separated using a chiral HPLC column. Alternatively, specific stereoisomers can be synthesized using an optically active starting material, by asymmetric synthesis using optically active reagents, substrates, catalysts, or solvents, or by converting one stereoisomer to another using an asymmetric transformation.

When compounds of the present invention contain two or more stereogenic centers and the absolute or relative stereochemistry is indicated in the name, the designations R and S refer, respectively, to each stereogenic center in ascending order of numbers (1, 2, 3, etc.) according to with generally accepted IUPAC numbering schemes for each molecule. When the compounds of the present invention contain one or more stereogenic centers and no stereochemistry is indicated in the name or structure, the name or structure is intended to cover all forms of the compound, including the racemic form.

It is also possible that the intermediates and compounds of the present invention may exist in various tautomeric forms, in which case all such forms are included within the scope of the present invention. The term "tautomer" or "tautomeric form" refers to structural isomers with different energies and a low energy barrier to their interconversion. For example, interconversions of protic tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerizations.

Interconversions of valency tautomers include interconversions through redistribution of some of the bonding electrons.

The scope of the claimed compounds of the present invention includes all stereoisomers, geometric isomers and tautomeric forms of the compounds of the present invention, including compounds exhibiting multiple types of isomerism and mixtures of isomers of one or more types. Also included within the scope of the present invention are acid addition or base salts in which the counterion is optically active, for example D-lactate or L-lysine or their racetams, for example DL-tartrate or DL-arginine.

The invention includes all pharmaceutically acceptable isotopically labeled compounds of the present invention in which one or more atoms are replaced by atoms of the same atomic number, but with an atomic mass or mass number different from the atomic mass or mass number that is commonly found in nature.

Examples of isotopes suitable for inclusion in the compounds of the present invention include isotopes of hydrogen such as ² H and ³ H, carbon such as ¹¹ C, ¹³ C and ¹⁴ C, chlorine such as ³⁶ Cl, fluorine such as ¹⁸ F , iodine such as ¹²³ I, ¹²⁴ I and ¹²⁵ I, nitrogen such as ¹³ N and ¹⁵ N, oxygen such as ¹⁵ O, ¹⁷ O and ¹⁸ O, phosphorus such as ³² P, and sulfur such as ³⁵ S.

Certain isotopically labeled compounds of the present invention, for example compounds containing a radioactive isotope, may be used in drug and/or substrate tissue distribution studies. Radioactive isotomes of tritium, i.e. ³ H, and carbon-14, i.e. ¹⁴ C are particularly useful for this purpose due to their ease of administration and off-the-shelf detection tools.

Substitution by heavier isotopes such as deuterium, i.e. ² H may lead to some therapeutic benefits that result from greater metabolic stability, such as an increase in in vivo half-life or a reduction in the required dose, and therefore may be preferred in some cases.

Substitution with positron-emitting isotopes such as ¹¹ C, ¹⁸ F, ¹⁵ O and ¹³ N can be used in positron emission tomography (PET) studies to determine the degree of substrate receptor occupancy.

Isotopically labeled compounds of the present invention may generally be prepared by standard methods known to those skilled in the art, or by methods similar to those described in the accompanying Examples and Preparation Examples, using appropriate isotopically labeled reagents in place of the unlabeled reagents previously used.

The compounds of the present invention may be isolated and used as such or, when possible, in the form of their pharmaceutically acceptable salts. The term "salts" refers to inorganic and organic salts of the compounds of the present invention. These salts may be obtained in situ during the final isolation and purification of the compound, or by separately treating the compound with a suitable organic or inorganic acid or organic or inorganic base to isolate the salt thus formed. The acids which are used to prepare the pharmaceutically acceptable acid addition salts of the aforementioned basic compounds of the present invention are acids which form non-toxic acid addition salts (i.e. salts containing pharmacologically acceptable anions such as hydrochloride, hydrobromide, hydroiodide , nitrate, sulfate, bisulfate, phosphate, hydrogen phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, naphthylate, mesylate, glucoheptonate, lactobionate , laurylsulfonate, hexafluorophosphate, benzenesulfonate, tosylate, formate, trifluoroacetate, oxalate, besylate, palmitate, pamoate, malonate, stearate, laurate, malate, borate, p-toluenesulfonate and pamoate (i.e. 1,1'-methylene-bis- (2-hydroxy-3-naphthoate)).

The invention also relates to base addition salts of the compounds of the present invention. Chemical bases that can be used as reagents for preparing pharmaceutically acceptable base salts of the compounds of the present invention that are acidic in nature are bases which form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to, salts derived from pharmacologically acceptable cations such as alkali metal (eg, lithium, potassium, and sodium) cations and alkaline earth metal (eg, calcium and magnesium) cations, ammonium salts, or water-soluble amino addition salts such as N-methylglucamine (meglumine), tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine and lower alkanolammonium and other basic salts of pharmacologically acceptable organic amines (see, for example, Berge, et al J Pharm Sci 66 1-19 (1977).

Some compounds of the present invention may exist in multiple crystalline forms (commonly referred to as "polymorphs"). Polymorphs can be obtained by crystallization under various conditions, for example using different solvents or different mixtures of recrystallization solvents; crystallization at various temperatures; and/or with various cooling methods - from very fast to very slow cooling during crystallization. Polymorphs can also be obtained by heating or melting the compound of the present invention, followed by gradual or rapid cooling. The presence of polymorphs can be determined using solid state NMR spectroscopy, IR spectroscopy, differential scanning calorimetry, X-ray powder diffraction, or other such methods.

In one embodiment, the present invention relates to a pharmaceutical composition comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine -5-carboxamide or a pharmaceutically acceptable salt thereof in combination with at least 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1 '-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, each of these compounds in a therapeutically effective amount, in admixture with a pharmaceutically acceptable excipient.

In another embodiment of the present invention, the composition further comprises at least one additional pharmaceutical agent selected from the group consisting of an anti-inflammatory agent, an antidiabetic agent, and a cholesterol/lipid modulating agent.

In another embodiment of the present invention, the composition also includes at least one additional pharmaceutical agent selected from the group consisting of [(1R,5S,6R)-3-{2-[(2S)-2-methylazetidin-1-yl] -6-(trifluoromethyl)pyrimidin-4-yl}-3-azabicyclo[3.1.0]hex-6-yl]acetic acid; 2-[(1R,3R,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1 ]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acid; or 2-[(4-{6-[(4-cyano-2-fluorobenzyl)oxy]pyridin-2-yl}piperidin-1-yl)methyl]-1-[(2S)-oxetan-2-ylmethyl] -1H-benzimidazole-6-carboxylic acid, or a pharmaceutically acceptable salt thereof.

In another embodiment of the present invention, the pharmaceutical composition comprises (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5- carboxamide in the form of a crystalline solid structure:

or a pharmaceutically acceptable salt thereof.

In another embodiment of the present invention, the powder X-ray diffraction pattern of said crystalline solid contains peaks at 2-theta angle (CuKα radiation, wavelength 1.54056 Å) of 5.3 ± 0.2, 7.7 ± 0.2 and 15, 4 ± 0.2.

In another embodiment of the present invention, the X-ray powder diffraction pattern of said crystalline solid contains peaks at 2-theta angle (CuKα radiation, wavelength 1.54056Å) of 6.5 ± 0.2, 9.3 ± 0.2 and 13.6 ±0.2.

In yet another embodiment of the present invention, the pharmaceutical composition contains 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)- 6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof in the form of a crystalline solid with the structure:

or a pharmaceutically acceptable salt thereof.

In yet another embodiment of the present invention, the crystalline solid is 2-amino-2-(hydroxymethyl)propane-1,3-diol salt 4-(4-(1-isopropyl-7-oxo-1,4,6,7 -tetrahydrospiro[indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid.

In yet another embodiment of the present invention, the pharmaceutical composition also includes at least one additional pharmaceutical agent selected from the group consisting of [(1R,5S,6R)-3-{2-[(2S)-2-methylazetidine-1- yl]-6-(trifluoromethyl)pyrimidin-4-yl}-3-azabicyclo[3.1.0]hex-6-yl]acetic acid; 2-[(1R,3R,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1 ]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acid; or 2-[(4-{6-[(4-cyano-2-fluorobenzyl)oxy]pyridin-2-yl}piperidin-1-yl)methyl]-1-[(2S)-oxetan-2-ylmethyl] -1H-benzimidazole-6-carboxylic acid, or a pharmaceutically acceptable salt thereof.

In yet another embodiment, the present invention relates to a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver, and non-alcoholic steatohepatitis with cirrhosis of the liver and hepatocellular carcinoma or with a metabolic disease, said method comprising administering to a patient in need of such treatment a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N- (tetrahydrofuran-3-yl)pyrimidine-5-carboxamide, or a pharmaceutically acceptable salt thereof, in combination with at least a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[ indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)-benzoic acid or a pharmaceutically acceptable salt thereof.

In yet another embodiment of the present invention, the disease or condition is a fatty liver. In another embodiment of the present invention, the disease or condition is non-alcoholic fatty liver disease. In yet another embodiment of the present invention, the disease or condition is non-alcoholic steatohepatitis. In yet another embodiment of the present invention, the disease or condition is non-alcoholic steatohepatitis with liver fibrosis. In yet another embodiment of the present invention, the disease or condition is non-alcoholic steatohepatitis with cirrhosis of the liver. In yet another embodiment of the present invention, the disease or condition is non-alcoholic steatohepatitis with cirrhosis of the liver and with hepatocellular carcinoma. In yet another embodiment of the present invention, the disease or condition is non-alcoholic steatohepatitis with cirrhosis of the liver and metabolic disease.

In yet another embodiment of the present invention, the method comprises at least one other pharmaceutical agent, wherein said pharmaceutical agent is selected from the group consisting of an acetyl-CoA carboxylase (ACC) inhibitor, a diacylglycerol-O-acyltransferase 1 (DGAT-1) inhibitor, monoacylglycerol-O-acyltransferase inhibitors, phosphodiesterase-10 inhibitor (phosphodiesterase 10 - PDE-10), AMPK activator, sulfonylurea, meglitinide, α-amylase inhibitor, α-glucoside hydrolase inhibitor, α-glucosidase inhibitor, PPARγ agonist, PPAR α/γ agonist , biguanide, modulator of glucagon-like peptide 1 (glucagon-like peptide 1 - GLP-1), liraglutide, albiglutide, exenatide, albiglutide, lixisenatide, dulaglutide, semaglutide, protein tyrosine phosphatase-1B inhibitor (protein tyrosine phosphatase-1B - PTP-1B), activator SIRT-1, dipeptidyl peptidase IV (DPP-IV) inhibitor, insulin secretagogue, fatty acid oxidation inhibitor, A2 antagonist, c-Jun N-t inhibitor terminal kinase (JNK), glucokinase (GKa) activators, insulin, insulin like agent, glycogen phosphorylase inhibitor, VPAC2t receptor agonist, SGLT2 inhibitors, glucagon receptor modulator, GPR119 modulators, F21 derivatives and analogs, TGR5 receptor modulators, GPBAR1 receptor modulators, GPR40 agonists, GPR120 modulators, high affinity nicotinic acid receptor (HM74A) activators, SGLT1 inhibitors, carnitine palmitoyltransferase enzyme inhibitors or modulators, fructose 1,6-diphosphatase inhibitors, aldose reductase inhibitors, mineralocorticoid receptor inhibitors, TORC2 inhibitors, CCR2 and/or CCR5 inhibitors, isoform inhibitors PKC (eg, PKCα, PKCβ, PKCγ), fatty acid synthetase inhibitors, serine palmitoyl transferase inhibitors, GPR81, GPR39, GPR43, GPR41, GPR105, Kv1.3 modulators, retinol-binding protein-4, glucocorticoid receptor, somatostatin receptors, PDHK2 inhibitors or modulators, or PDHK4, MAP4 inhibitors K4, IL1 family modulators including IL1beta, hydroxymethylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitors, squalene synthetase inhibitors, fibrates, bile acid sequestrants, ACAT inhibitors, MTP inhibitors, lipooxygenase inhibitors, cholesterol absorption inhibitors, PCSK9 modulators, transport inhibitors cholesteryl ester protein and RXRalpha modulators.

In yet another embodiment of the present invention, the method comprises at least one other pharmaceutical selected from the group consisting of cysteamine or a pharmaceutically acceptable salt thereof, cystamine or a pharmaceutically acceptable salt thereof, an antioxidant compound, lecithin, a vitamin B complex, bile salt preparations, cannabinoid receptor 1 (Cannabinoid-1 - CB1) antagonists, cannabinoid 1 (CB1) receptor inverse antagonists, regulators of the activity of peroxisome proliferator-activated receptors, a benzothiazepine or benzothiepine derivative, an antisense RNA construct for the inhibition of PTPRU protein tyrosine phosphatase, piperidine linked through a heteroatom, and its derivatives, an azacyclopentane derivative capable of inhibiting stearoyl-coenzyme alpha-delta-9-desaturase, an acylamide compound having the activity of a secretion stimulant or an inducer of adiponectin activity, a quaternary ammonium compound, glatiramer acetate, penta traxin proteins, an HMG-CoA reductase inhibitor, n-acetylcysteine, an isoflavone compound, a macrolide antibiotic, a galectin inhibitor, an antibody, or any combination thereof.

In yet another embodiment of the present invention, the method includes at least one other pharmaceutical agent, where the specified pharmaceutical agent is selected from the group consisting of [(1R,5S,6R)-3-{2-[(2S)-2-methylazetidine-1 -yl]-6-(trifluoromethyl)pyrimidin-4-yl}-3-azabicyclo[3.1.0]hex-6-yl]acetic acid; 2-[(1R,3R,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1 ]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acid; or 2-[(4-{6-[(4-cyano-2-fluorobenzyl)oxy]pyridin-2-yl}piperidin-1-yl)methyl]-1-[(2S)-oxetan-2-ylmethyl] -1H-benzimidazole-6-carboxylic acid, or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention also relates to a method for reducing at least one point in a scoring system for the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, reducing the level of serum markers of non-alcoholic steatohepatitis activity, reducing the disease activity of non-alcoholic steatohepatitis, or reducing the medical consequences of non-alcoholic steatohepatitis in humans, comprising the step of administering to a patient in need of such a reduction a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3- yl)pyrimidine-5-carboxamide, or a pharmaceutically acceptable salt thereof, in combination with at least a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4 '-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)-benzoic acid or a pharmaceutically acceptable salt thereof.

In yet another embodiment of the present invention, the method includes at least one other pharmaceutical agent, where the specified pharmaceutical agent is selected from the group consisting of [(1R,5S,6R)-3-{2-[(2S)-2-methylazetidine- 1-yl]-6-(trifluoromethyl)pyrimidin-4-yl}-3-azabicyclo[3.1.0]hex-6-yl]acetic acid; 2-[(1R,3R,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1 ]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acid; or 2-[(4-{6-[(4-cyano-2-fluorobenzyl)oxy]pyridin-2-yl}piperidin-1-yl)methyl]-1-[(2S)-oxetan-2-ylmethyl] -1H-benzimidazole-6-carboxylic acid, or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention relates to a method of treating fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver, or non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma in humans, comprising the step of administering to a person in need of such treatment, a therapeutically effective amount of two separate pharmaceutical compositions, including

i. a first composition which comprises (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide or a pharmaceutically an acceptable salt in a therapeutically effective amount in admixture with a pharmaceutically acceptable excipient;

ii. the second composition, which includes 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridine-2 -yl)benzoic acid or a pharmaceutically acceptable salt thereof in a therapeutically effective amount in admixture with a pharmaceutically acceptable excipient; and optionally

iii. a third composition comprising at least one pharmaceutical agent selected from the group consisting of an anti-inflammatory agent, an antidiabetic agent, an antifibrotic agent, an antisteatotic agent, a cholesterol/lipid modulating agent, and an antidiabetic agent, and a pharmaceutically acceptable excipient.

In yet another embodiment of the present invention, said first composition and said second composition are administered simultaneously. In another embodiment of the present invention, the composition includes a first composition, a second composition and a third composition.

In yet another embodiment of the present invention, the method includes a third composition wherein at least one other pharmaceutical agent is selected from the group consisting of [(1R,5S,6R)-3-{2-[(2S)-2-methylazetidine- 1-yl]-6-(trifluoromethyl)pyrimidin-4-yl}-3-azabicyclo[3.1.0]hex-6-yl]acetic acid; 2-[(1R,3R,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1 ]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acid; or 2-[(4-{6-[(4-cyano-2-fluorobenzyl)oxy]pyridin-2-yl}piperidin-1-yl)methyl]-1-[(2S)-oxetan-2-ylmethyl] -1H-benzimidazole-6-carboxylic acid or their pharmaceutically acceptable salts.

In yet another embodiment of the present invention, (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof in combination with 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridine -2-yl)benzoic acid or a pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in a therapeutically effective amount in admixture with a pharmaceutically acceptable excipient.

In yet another embodiment of the present invention, (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide or its pharmaceutically acceptable salt in combination with at least 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)- 6-Methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in a therapeutically effective amount in admixture with a pharmaceutically acceptable excipient.

In yet another embodiment of the present invention, the composition also comprises at least one additional pharmaceutical agent selected from the group consisting of a GLP-1R agonist, a KHK inhibitor, an FXR agonist, an anti-inflammatory agent, an antidiabetic agent, an antifibrotic agent, an antisteatotic agent, and a cholesterol/lipid - modulating agent.

In yet another embodiment of the present invention, a method of treating a metabolic disease, condition, or disorder, or a metabolic disease, condition, or disorder, comprises the step of administering to a patient a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridine-2- yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof in combination with 4-(4-(1-isopropyl-7-oxo-1,4 ,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof.

In yet another embodiment of the present invention, a method of treating a condition selected from the group consisting of hyperlipidemia, type I diabetes, type II diabetes mellitus, type I idiopathic diabetes (type Ib), latent adult autoimmune diabetes (LADA), juvenile diabetes (EOD) Type 2 diabetes, juvenile atypical diabetes (YOAD), juvenile adult-onset diabetes (MODY), malnutrition-related diabetes, gestational diabetes, coronary heart disease, ischemic stroke, angioplasty restenosis, peripheral vascular disease, intermittent claudication, myocardial infarction ( eg, necrosis and apoptosis), dyslipidemia, postprandial lipemia, conditions of impaired glucose tolerance (IGT), conditions of impaired fasting plasma glucose, metabolic acidosis, ketosis, arthritis, obesity, osteoporosis, hypertension, congestive heart failure, left ventricular hypertrophy , peripheral arterial disease, diabetic severe retinopathy, macular degeneration, cataracts, diabetic nephropathy, glomerulosclerosis, chronic renal failure, diabetic neuropathy, metabolic syndrome, syndrome X, premenstrual syndrome, coronary heart disease, angina pectoris, thrombosis, atherosclerosis, myocardial infarction, transient ischemic attacks, stroke, vascular restenosis, hyperglycemia, hyperinsulinemia, hyperlipidemia, hypertriglyceridemia, insulin resistance, disorders of glucose metabolism, conditions of impaired glucose tolerance, conditions of impaired fasting glucose, obesity, erectile dysfunction, skin and connective tissue disorders, foot ulcers and ulcerative colitis, endothelial dysfunction and elasticity disorders vascular disease, hyperapolipoproteinemia B, Alzheimer's disease, schizophrenia, cognitive impairment, inflammatory bowel disease, ulcerative colitis, Crohn's disease and irritable bowel syndrome, non-alcoholic steatohepatitis a (NASH), non-alcoholic fatty liver disease (NAFLD), comprises administering a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of Compound D or a pharmaceutically acceptable salt thereof.

In yet another embodiment of the present invention, a method of treating a metabolic disease, condition, or disorder, or a metabolic disease, condition, or disorder, comprises the step of administering to a patient in need of such treatment at least two separate pharmaceutical compositions, including

(i) a first composition which comprises (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide or a pharmaceutically acceptable salt thereof in a therapeutically effective amount in admixture with a pharmaceutically acceptable excipient;

(ii) a second composition which comprises 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6- methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, in a therapeutically effective amount, in admixture with a pharmaceutically acceptable excipient; and optionally

(iii) a third composition comprising at least one pharmaceutical agent selected from the group consisting of a GLP-1R agonist, a KHK inhibitor, an FXR agonist, an anti-inflammatory agent, an antidiabetic agent, an antifibrotic agent, an antisteatotic agent, a cholesterol/lipid modulating agent, and an antidiabetic agent; and a pharmaceutically acceptable excipient.

In yet another embodiment of the present invention, the method of the present invention is carried out when said first composition, said second composition and said third composition are administered simultaneously.

In yet another embodiment of the present invention, the method of the present invention is carried out when said first composition, said second composition and said third composition are administered sequentially in any order.

In one embodiment of the present invention, when three pharmaceutical agents are administered, the first pharmaceutical agent and the second pharmaceutical agent are administered simultaneously, and the third pharmaceutical agent is administered sequentially. In another embodiment of the present invention, three separate pharmaceutical agents are administered sequentially in any order.

In one embodiment of the present invention, when three pharmaceutical agents are administered, the third pharmaceutical agent comprises a GLP-1R agonist. GLP-1R agonist 2-[(4-{6-[(4-cyano-2-fluorobenzyl)oxy]pyridin-2-yl}piperidin-1-yl)methyl]-1-[(2S)-oxetan- 2-ylmethyl]-1H-benzimidazole-6-carboxylic acid or a pharmaceutically acceptable salt thereof [such as its 2-amino-2-(hydroxymethyl)propan-1,3-diol salt, also known as its tris salt], and also other GLP-1R agonists and methods for preparing these compounds are described in US Patent No. 10208019, the contents of which are incorporated herein by reference for all purposes.

In some embodiments, the GLP-1R agonist is selected from the group consisting of liraglutide, albiglutide, exenatide, albiglutide, lixisenatide, dulaglutide, semaglutide, HM15211, LY3298176, Medi-0382, NN-9924, TTP-054, TTP-273, efpenglenatide, the agonists described in WO2018109607, and DIAST-X2.

GLP-1 is a 30 amino acid incretin hormone secreted by intestinal L cells in response to food intake. GLP-1 has been shown to stimulate insulin secretion in a physiological and glucose-dependent manner, decrease glucagon secretion, inhibit gastric emptying, reduce appetite, and stimulate beta cell proliferation. In non-clinical experiments, GLP-1 promotes continued beta cell competence by stimulating the transcription of genes important for glucose-dependent insulin secretion and by stimulating beta cell neogenesis (Meier, et al. Biodrugs. 2003; 17(2): 93-102).

In a healthy individual, GLP-1 plays an important role in the regulation of postprandial blood glucose levels by stimulating glucose-dependent insulin secretion from the pancreas, resulting in increased peripheral glucose uptake. GLP-1 also suppresses the secretion of glucagon, which leads to a decrease in glucose production in the liver. In addition, GLP-1 delays gastric emptying and slows down peristalsis of the small intestine, delaying food absorption. In people with T2DM, the normal increase in GLP-1 levels after a meal is absent or reduced (Vilsboll T., et al. Diabetes. 2001. 50; 609-613).

Holst (Physiol. Rev. 2007, 87, 1409) and Meier (Nat. Rev. Endocrinol. 2012, 8, 728) report that GLP-1 receptor agonists such as GLP-1, liraglutide and exendin-4 have three main pharmacological activities to improve glycemic control in patients, such as patients with T2DM, by reducing fasting and postprandial glucose (FPG and PPG) levels: (i) increased glucose-dependent insulin secretion (improved first and second phases), (ii) glucagon suppression activity in hyperglycemic states, (iii) slowing down the rate of gastric emptying resulting in delayed absorption of food-derived glucose.

In another embodiment of the present invention, when three pharmaceutical agents are administered, the third agent comprises a KHK inhibitor.

In some embodiments, the KHK inhibitor is [(1R,5S,6R)-3-{2-[(2S)-2-methylazetidin-1-yl]-6-(trifluoromethyl)pyrimidin-4-yl}- 3-azabicyclo[3.1.0]hex-6-yl]acetic acid or pharmaceutically acceptable salts thereof. [(1R,5S,6R)-3-{2-[(2S)-2-methylazetidin-1-yl]-6-(trifluoromethyl)pyrimidin-4-yl}-3-azabicyclo[3.1.0]hex- 6-yl]acetic acid (including its free acid crystalline form) is a ketohexokinase inhibitor and is described in Example 4 of US Pat. No. 9,809,579, the contents of which are incorporated herein by reference in its entirety for all purposes.

In some embodiments, the KHK inhibitor is the crystalline form of the free [(1R,5S,6R)-3-{2-[(2S)-2-methylazetidin-1-yl]-6-(trifluoromethyl)pyrimidine-4- yl}-3-azabicyclo[3.1.0]hex-6-yl]acetic acid.

Ketohexokinase (KHK) is a key enzyme in fructose metabolism and catalyzes the conversion of fructose to fructose-1-phosphate (F1P). KHK is expressed as two alternative mRNA splice variants, designated KHKa and KHKc, resulting from alternative third exon splicing. The affinity and ability to phosphorylate fructose in KHKc is significantly higher than in KHKa, as evidenced by the much lower Km (Ishimoto, Lanaspa et al., PNAS 109, 4320-4325, 2012). Although KHKa is ubiquitously expressed, KHKc expression is highest in the liver, kidney, and intestine, major sites of fructose metabolism in the body (Diggle C.P., et al. (2009) J. Histochem. Cytochem. 57:763-774; Ishimoto, Lanaspa, et al. ., PNAS 109, 4320-4325, 2012). A fructose loss-of-function mutation has also been described in humans without any side effects, except for the appearance of fructose in the urine after sugar ingestion, this mutation is called essential fructosuria (OMIM #229800).

A more severe condition associated with fructose metabolism is Hereditary Fructose Intolerance (HFI, OMIM #229600), which is due to defects in aldolase B (GENE: ALDOB), the enzyme responsible for the breakdown of F1P and located just below the KHK stage in metabolism. (Bouteldja N., et. al, J. Inherit. Metab. Dis. 2010 Apr;33(2):105-12; Tolan, D.R., Hum. Mutat. 1995;6(3):210-8; http: //www.omim.org/entry/229600). It is a rare disorder affecting approximately 1 in 20,000 people, and these mutations lead to F1P accumulation, ATP depletion, and elevated uric acid levels, leading to hypoglycemia, hyperuricemia, and lactose, among other metabolic disorders. HFI interferes with the body's ability to metabolize dietary fructose, causing acute symptoms such as vomiting, severe hypoglycemia, diarrhea, and abdominal upset leading to long-term growth failure, liver and kidney damage, and potential death (Ali M. et al., J Med Genet 1998 May:35(5):353-65). Patients usually suffer during the first years of life before diagnosis, and the only course of treatment is the elimination of fructose from the diet. Treatment is complicated by the presence of this macronutrient in most foods. In addition to physical symptoms, many patients feel emotionally and socially isolated as a consequence of their unusual diet and constantly monitor adherence to strict dietary restrictions (HFI-INFO Discussion Board, http://hfiinfo.proboards.com. Accessed 14 December 2015). Even when they appear asymptomatic, some patients develop NAFLD and kidney disease, highlighting the inadequacy of voluntary dietary restriction as the only treatment option and the high need for medical treatments for this condition.

In hyperglycemic conditions, endogenous glucose production occurs via the polyol metabolic pathway, whereby glucose is converted to fructose with sorbitol as an intermediate. The activity of this pathway is increased in hyperglycemia. In these studies, the authors demonstrated that KHK-null mice were protected from glucose-induced weight gain, insulin resistance, and hepatic steatosis, and suggested that in hyperglycemic states, endogenously produced fructose may contribute to the development of insulin resistance and hepatic steatosis (Lanaspa, M.A., et al., Nature Comm. 4, 2434, 2013). Therefore, KHK inhibition is expected to be beneficial in many diseases involving endogenous or dietary fructose or both.

In another embodiment of the present invention, when three pharmaceutical agents are administered, the third pharmaceutical agent comprises an FXR agonist. In some embodiments, the FXR agonist is selected from the group consisting of tropifexor (2-[(1R,3R,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2- oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acid) (“tropifexor”); cilofexor (GS-9674); obeticholic acid; LY2562175; Met409; TERN-101; and EDP-305 and their pharmaceutically acceptable salts. The FXR agonist tropifexor, or a pharmaceutically acceptable salt thereof, is described, for example, in Example 1-1B of US Patent No. 9,150,568, the contents of which are incorporated herein by reference in their entirety for all purposes. The chemical name of Tropifexor is 2-[(1R,3R,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo [3.2.1]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acid.

The farnesoid X receptor (FXR) is a member of the nuclear hormone superfamily and is expressed primarily in the oven, kidney, and intestine (see, for example, Seol et al. (1995) Mol. Endocrinol. 9:72-85; and Forman et al (1995) Cell 81:687-693). It functions as a heterodimer with the retinoid X receptor (RXR) and binds to response elements in promoters of target genes to regulate gene transcription. The FXR-RXR heterodimer binds with the highest affinity to the inverted repeat-1 (IR-1) response element, in which the receptor-binding consensus hexamers are separated by a single nucleotide. FXR is part of an interrelated process in which FXR is activated by bile acids (end products of cholesterol metabolism) (see e.g. Makishima et al. (1999) Science 284:1362-1365, Parks et al. (1999) Science 284:1365- 1368, Wang et al. (1999) Mol. Cell. 3:543-553), which serve to inhibit cholesterol catabolism (see also Urizar et al. (2000) J. Biol. Chem. 275:39313-39317).

FXR is a key regulator of cholesterol homeostasis, triglyceride synthesis and lipogenesis (Crawley, Expert Opinion Ther. Patents (2010), 20(8): 1047-1057). In addition to the treatment of dyslipidemia, a variety of indications for FXR have been described, including the treatment of liver disease, diabetes, vitamin D-related diseases, drug-induced side effects, and hepatitis (Crawley, supra). Despite advances in the development of new FXR agonists, significant room for improvement remains.

In some other embodiments, when three pharmaceutical agents are administered, the third pharmaceutical agent comprises an SGLT2 inhibitor, metformin, incretin analogs, an incretin receptor modulator, a DPP-4 inhibitor, or a PPAR agonist.

In some other embodiments, when three pharmaceutical agents are administered, the third pharmaceutical agent is an antidiabetic agent selected from metformin, sitagliptin, or ertugliflozin.

In some other embodiments, when three pharmaceuticals are administered, the third pharmaceutical is a heart failure agent selected from an ACE inhibitor, an angiotensin receptor blocker, a calcium channel blocker, or a vasodilator.

The present invention further includes kits that are suitable for use in the implementation of the methods of treatment described above. In one embodiment of the present invention, the kit contains a first dosage form comprising one or more drugs (compounds), or their pharmaceutically acceptable salts of the present invention and a dosage form container, in amounts sufficient to carry out the methods of the present invention.

In another embodiment of the present invention, the kit contains a first dosage form comprising one of the pharmaceutical agents (compounds) or a pharmaceutically acceptable salt thereof according to the present invention, and a container for the first dosage form, and a second dosage form comprising another pharmaceutical agent (compound) or its a pharmaceutically acceptable salt of the present invention; and a container for a second dosage form, wherein both dosage forms are present in amounts sufficient to carry out the methods of the present invention.

In another embodiment of the present invention, the kit contains a first dosage form comprising one of the pharmaceutical agents (compounds) or a pharmaceutically acceptable salt thereof according to the present invention, and a container for the first dosage form, a second dosage form comprising another pharmaceutical agent (compound) or its pharmaceutically acceptable an acceptable salt of the present invention, and a container for a second dosage form and a third dosage form comprising another pharmaceutical agent (compound) or a pharmaceutically acceptable salt thereof of the present invention, and a container for a third dosage form, where all three dosage forms are present in amounts, sufficient to carry out the methods of the present invention.

In some embodiments, the present invention provides a kit that includes a first dosage form comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran- 3-yl)pyrimidin-5-carboxamide or a pharmaceutically acceptable salt thereof, a first dosage form container and a second dosage form comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[ indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, and a container for a second dosage form, wherein both dosage forms are present in amounts sufficient to carry out methods of the present invention.

In another embodiment, the present invention provides a kit that includes a first dosage form comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran- 3-yl)pyrimidin-5-carboxamide or a pharmaceutically acceptable salt thereof, a container for a first dosage form, a second dosage form comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[ indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, a second dosage form container and a third dosage form comprising another drug (compound) or a pharmaceutically acceptable salt thereof; and a container for a third dosage form wherein all three dosage forms are present in amounts sufficient to carry out the methods of the present invention.

In some embodiments, the dosage form(s) of the kit may be administered simultaneously or chronologically delayed, that is, at different points in time and at equal or different time intervals for any of the dosage forms of the kit.

In any of the kits described above, means are provided for storing each dosage form separately, such as a container, a split vial, or a split foil pouch. An example of such a kit is the known blister pack used for filling tablets, capsules and the like. Such a kit is particularly suitable for administering different dosage forms, for example oral and parenteral dosage forms, for administering individual drugs (compounds) at different dosing intervals, or for titration of a single drug (compound) against another. To comply with the therapy regimen, the kit may include instructions for administration and may be provided with a so-called reminder.

The present invention also includes various other kits used for the packaging, distribution and administration of the pharmaceutical agents (compounds) of the present invention. In yet another embodiment, the present invention relates to a pharmaceutical composition comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'- carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, an HMG-CoA reductase inhibitor and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl )-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, an HMG-CoA reductase inhibitor selected from the group consisting of pravastatin, pitavastatin, lovastatin, atorvastatin, simvastatin, fluvastatin, itavastatin, nisvastatin, nisbastatin, rosuvastatin, atavastatin and visastatin; and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl )-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, atorvastatin and a pharmaceutically acceptable excipient.

In yet another embodiment, the present invention relates to a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver, and non-alcoholic steatohepatitis with cirrhosis of the liver and hepatocellular carcinoma or with a metabolic disease, said method comprising administering to a person in need of such treatment a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4' -piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof and an HMG-CoA reductase inhibitor. In some embodiments, the HMG-CoA reductase inhibitor is atorvastatin.

In yet another embodiment, the present invention relates to a method for reducing at least one point in a scoring system for the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, reducing the level of serum markers of non-alcoholic steatohepatitis activity, reducing the disease activity of non-alcoholic steatohepatitis, or reducing the medical consequences of non-alcoholic steatohepatitis in humans, comprising the step of administering to a patient in need of such a reduction a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1 '-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof and an HMG-CoA reductase inhibitor. In some embodiments, the HMG-CoA reductase inhibitor is atorvastatin.

In yet another embodiment, the present invention relates to a pharmaceutical composition comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'- carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, a fibrate agent and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl )-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, a fibrate agent selected from the group consisting of fibrozil, fenofibrate and clofibrate, and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl )-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, fenofibrate and a pharmaceutically acceptable excipient.

In yet another embodiment, the present invention relates to a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver, and non-alcoholic steatohepatitis with cirrhosis of the liver and hepatocellular carcinoma or with a metabolic disease, said method comprising administering to a person in need of such treatment a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4' -piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof and a fibrate agent. In some embodiments, the fibrate agent is fenofibrate.

In a further embodiment, the present invention relates to a method for reducing at least one point in scoring systems for the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, reducing serum markers of non-alcoholic steatohepatitis activity, reducing disease activity of non-alcoholic steatohepatitis, or reducing the medical consequences of non-alcoholic steatohepatitis in humans. comprising the step of administering to a patient in need of such a reduction a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1' -carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof and a fibrate agent. In some embodiments, the fibrate agent is fenofibrate.

In yet another embodiment, the present invention relates to a pharmaceutical composition comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'- carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, a bile acid sequestrant and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl )-6-methoxypyridin-2-yl)-benzoic acid or a pharmaceutically acceptable salt thereof, a bile acid sequestrant selected from the group consisting of questran, colestipol and colesevelam, and a pharmaceutically acceptable excipient.

In yet another embodiment, the present invention relates to a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver, and non-alcoholic steatohepatitis with cirrhosis of the liver and hepatocellular carcinoma or with a metabolic disease, said method comprising administering to a person in need of such treatment a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4' -piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof and a bile acid sequestrant.

In yet another embodiment, the present invention relates to a method for reducing at least one point in a scoring system for the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, reducing the level of serum markers of non-alcoholic steatohepatitis activity, reducing the disease activity of non-alcoholic steatohepatitis, or reducing the medical consequences of non-alcoholic steatohepatitis in humans, comprising the step of administering to a patient in need of such a reduction a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1 '-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof and a bile acid sequestrant.

In yet another embodiment, the present invention relates to a pharmaceutical composition comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'- carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, a cholesterol absorption inhibitor and a pharmaceutically acceptable excipient. In some embodiments, the cholesterol absorption inhibitor is ezetimibe.

In yet another embodiment, the present invention relates to a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver, and non-alcoholic steatohepatitis with cirrhosis of the liver and hepatocellular carcinoma or with a metabolic disease, said method comprising administering to a person in need of such treatment a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4' -piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof; and a cholesterol absorption inhibitor.

In yet another embodiment, the present invention relates to a method for reducing at least one point in non-alcoholic fatty liver disease or non-alcoholic steatohepatitis severity score systems, lowering serum markers of non-alcoholic steatohepatitis activity, reducing non-alcoholic steatohepatitis disease activity, or reducing the medical consequences of non-alcoholic steatohepatitis in humans. comprising the step of administering to a patient in need of such a reduction a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1' -carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof; and a cholesterol absorption inhibitor.

In yet another embodiment, the present invention relates to a pharmaceutical composition comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'- carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, a nicotinic acid pharmaceutical agent and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl )-6-methoxypyridin-2-yl)-benzoic acid or a pharmaceutically acceptable salt thereof, a nicotinic acid pharmaceutical agent selected from the group consisting of niacin, niacor and slo-niacin, and a pharmaceutically acceptable excipient.

In yet another embodiment, the present invention relates to a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver, and non-alcoholic steatohepatitis with cirrhosis of the liver and hepatocellular carcinoma or with a metabolic disease, said method comprising administering to a person in need of such treatment a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4' -piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof; and a nicotinic acid pharmaceutical agent.

In yet another embodiment, the present invention relates to a method for reducing at least one point in a scoring system for the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, reducing the level of serum markers of non-alcoholic steatohepatitis activity, reducing the disease activity of non-alcoholic steatohepatitis, or reducing the medical consequences of non-alcoholic steatohepatitis in humans, comprising the step of administering to a patient in need of such a reduction a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1 '-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof; and a nicotinic acid pharmaceutical agent.

In yet another embodiment, the present invention relates to a pharmaceutical composition comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'- carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, a PCSK9 modulator and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl )-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, a PCSK9 modulator selected from the group consisting of alirocumab and evolucumab, and a pharmaceutically acceptable excipient.

In yet another embodiment, the present invention relates to a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver, and non-alcoholic steatohepatitis with cirrhosis of the liver and hepatocellular carcinoma or with a metabolic disease, said method comprising administering to a person in need of such treatment a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4' -piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof; and a PCSK9 modulator.

In yet another embodiment, the present invention relates to a method for reducing at least one point in a scoring system for the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, reducing the level of serum markers of non-alcoholic steatohepatitis activity, reducing the disease activity of non-alcoholic steatohepatitis, or reducing the medical consequences of non-alcoholic steatohepatitis in humans, comprising the step of administering to a patient in need of such a reduction a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1 '-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof and a PCSK9 modulator.

In any of the previous embodiments, 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridine- 2-yl)benzoic acid is a crystalline solid. In some embodiments, the crystalline solid is 2-amino-2-(hydroxymethyl)propane-1,3-diol salt 4-(4-(1-isopropyl-7-oxo-1,4,6,7- tetrahydrospiro[indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid.

In another embodiment, the present invention relates to a pharmaceutical composition comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl) pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof, an HMG-CoA reductase inhibitor and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine -5-carboxamide or a pharmaceutically acceptable salt thereof, an HMG-CoA reductase inhibitor selected from the group consisting of pravastatin, pitavastatin, lovastatin, atorvastatin, simvastatin, fluvastatin, itavastatin, nisvastatin, nisbastatin, rosuvastatin, atavastatin and visastatin, and a pharmaceutically acceptable excipient .

In some embodiments, the present invention relates to a pharmaceutical composition comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine -5-carboxamide or a pharmaceutically acceptable salt thereof, atorvastatin and a pharmaceutically acceptable excipient.

In yet another embodiment, the present invention relates to a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver, and non-alcoholic steatohepatitis with cirrhosis of the liver and hepatocellular carcinoma or with a metabolic disease, said method comprising administering to a person in need of such treatment a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N- (tetrahydrofuran-3-yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof and an HMG-CoA reductase inhibitor. In some embodiments, the HMG-CoA reductase inhibitor is atorvastatin.

In yet another embodiment, the present invention relates to a method for reducing at least one point in a scoring system for the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, reducing the level of serum markers of non-alcoholic steatohepatitis activity, reducing the disease activity of non-alcoholic steatohepatitis, or reducing the medical consequences of non-alcoholic steatohepatitis in humans, comprising the step of administering to a patient in need of such a reduction a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3- yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof and an HMG-CoA reductase inhibitor. In some embodiments, the HMG-CoA reductase inhibitor is atorvastatin.

In yet another embodiment, the present invention relates to a pharmaceutical composition comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl) pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof, a fibrate agent and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine -5-carboxamide or a pharmaceutically acceptable salt thereof, a fibrate agent selected from the group consisting of gemfibrozil, fenofibrate and clofibrate, and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine -5-carboxamide or a pharmaceutically acceptable salt thereof, fenofibrate and a pharmaceutically acceptable excipient.

In yet another embodiment, the present invention relates to a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver, and non-alcoholic steatohepatitis with cirrhosis of the liver and hepatocellular carcinoma or with a metabolic disease, said method comprising administering to a person in need of such treatment a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N- (tetrahydrofuran-3-yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof and a fibrate agent. In some embodiments, the fibrate agent is fenofibrate.

In yet another embodiment, the present invention relates to a method for reducing at least one point in a scoring system for the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, reducing the level of serum markers of non-alcoholic steatohepatitis activity, reducing the disease activity of non-alcoholic steatohepatitis, or reducing the medical consequences of non-alcoholic steatohepatitis in humans, comprising the step of administering to a patient in need of such a reduction a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3- yl) pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof and a fibrate agent. In some embodiments of the present invention, the fibrate agent is fenofibrate.

In yet another embodiment, the present invention relates to a pharmaceutical composition comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl) pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof, a bile acid sequestrant and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine -5-carboxamide or a pharmaceutically acceptable salt thereof, a bile acid sequestrant selected from the group consisting of questran, colestipol and colesevelam, and a pharmaceutically acceptable excipient.

In yet another embodiment, the present invention relates to a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver, and non-alcoholic steatohepatitis with cirrhosis of the liver and hepatocellular carcinoma or with a metabolic disease, said method comprising administering to a person in need of such treatment a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N- (tetrahydrofuran-3-yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof; and a bile acid sequestrant.

In yet another embodiment, the present invention relates to a method for reducing at least one point in a scoring system for the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, reducing the level of serum markers of non-alcoholic steatohepatitis activity, reducing the disease activity of non-alcoholic steatohepatitis, or reducing the medical consequences of non-alcoholic steatohepatitis in humans, comprising the step of administering to a human in need of such a reduction a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3- yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof and a bile acid sequestrant.

In another embodiment, the present invention relates to a pharmaceutical composition comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl) pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof, a cholesterol absorption inhibitor and a pharmaceutically acceptable excipient. In some embodiments, the cholesterol absorption inhibitor is ezetimibe.

In yet another embodiment, the present invention relates to a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver, and non-alcoholic steatohepatitis with cirrhosis of the liver and hepatocellular carcinoma or with a metabolic disease, said method comprising administering to a person in need of such treatment a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N- (tetrahydrofuran-3-yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof; and a cholesterol absorption inhibitor.

In yet another embodiment, the present invention relates to a method for reducing at least one point in a scoring system for the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, reducing the level of serum markers of non-alcoholic steatohepatitis activity, reducing the disease activity of non-alcoholic steatohepatitis, or reducing the medical consequences of non-alcoholic steatohepatitis in humans, comprising the step of administering to a patient in need of such a reduction a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3- yl) pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof; and a cholesterol absorption inhibitor.

In yet another embodiment, the present invention relates to a pharmaceutical composition comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl) pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof, a nicotinic acid pharmaceutical and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine -5-carboxamide or a pharmaceutically acceptable salt thereof, a nicotinic acid pharmaceutical agent selected from the group consisting of niacin, niacor and slo-niacin, and a pharmaceutically acceptable excipient.

In yet another embodiment, the present invention relates to a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver, and non-alcoholic steatohepatitis with cirrhosis of the liver and hepatocellular carcinoma or with a metabolic disease, said method comprising administering to a person in need of such treatment a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N- (tetrahydrofuran-3-yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof; and a nicotinic acid pharmaceutical agent.

In yet another embodiment, the present invention relates to a method for reducing at least one point in a scoring system for the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, reducing the level of serum markers of non-alcoholic steatohepatitis activity, reducing the disease activity of non-alcoholic steatohepatitis, or reducing the medical consequences of non-alcoholic steatohepatitis in humans, comprising the step of administering to a patient in need of such a reduction a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3- yl) pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof; and a nicotinic acid pharmaceutical.

In another embodiment, the present invention relates to a pharmaceutical composition comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl) pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof, a PCSK9 modulator and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine -5-carboxamide or a pharmaceutically acceptable salt thereof, a PCSK9 modulator selected from the group consisting of alirocumab and evolucumab, and a pharmaceutically acceptable excipient.

In yet another embodiment, the present invention relates to a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver, and non-alcoholic steatohepatitis with cirrhosis of the liver and hepatocellular carcinoma or with a metabolic disease, said method comprising administering to a person in need of such treatment a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N- (tetrahydrofuran-3-yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof; and a PCSK9 modulator.

In yet another embodiment, the present invention relates to a method for reducing at least one point in a scoring system for the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, reducing the level of serum markers of non-alcoholic steatohepatitis activity, reducing the disease activity of non-alcoholic steatohepatitis, or reducing the medical consequences of non-alcoholic steatohepatitis in humans, comprising the step of administering to a patient in need of such a reduction a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3- yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof and a PCSK9 modulator.

In any of the previous embodiments of the present invention, (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide is a solid crystalline substance. In some embodiments of the present invention, the X-ray powder diffraction pattern of said crystalline solid includes peaks at 2-theta angle (CuKα radiation, wavelength 1.54056 Å) of 5.3±0.2, 7.7±0.2, and 15, 4±0.2. In some other embodiments, the X-ray powder diffraction pattern of said crystalline solid includes peaks at 2-theta angle (CuKα radiation, wavelength 1.54056Å) of 6.5±0.2, 9.3±0.2, and 13.6± 0.2.

The compounds of the present invention can be synthesized by synthetic methods that include processes similar to those well known in the art, in particular those described herein. Starting materials are usually available from commercial sources such as Aldrich Chemicals (Milwaukee, WI) or prepared using methods well known to those skilled in the art (e.g., methods described in Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis , v. 1-19, Wiley, New York (1967-1999 ed.), or Beilsteins Handbuch der organischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin, including appendices (also available via the Beilstein online database) ). Preparation of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide is shown in Example 1 of the Patent Application US 2018-0051012A1, the contents of which are incorporated herein by reference for all purposes. Obtaining 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid is described in example 9 of US Patent No. 8859577, the contents of which are introduced into the present description by reference for all purposes. Preparation of [(1R,5S,6R)-3-{2-[(2S)-2-methylazetidin-1-yl]-6-(trifluoromethyl)pyrimidin-4-yl}-3-azabicyclo[3.1.0]hex -6-yl]acetic acid (including its free acid crystalline form) is described in Example 4 of US Patent No. 9809579. The preparation of GLP-1R agonists is described in US Patent No. 10208019.

PHARMACEUTICALS FOR COMBINED ADMINISTRATION

The compounds of the present invention may be administered alone or together as single pharmaceutical agents, in a fixed dose combination, or in combination with one or more additional therapeutic agents. The term "combination administration" or "combination therapy" means that Compound A and Compound D are administered together as two separate therapeutic agents or in combination with one or more additional therapeutic agents administered simultaneously to the mammal to be treated. When administered in combination, each component may be administered at the same time or sequentially in any order at different times. Therefore, each component can be administered separately, but at sufficiently close time points to provide the desired therapeutic effect. Thus, the methods of treatment described in the present invention include the use of combination agents to administer three or more agents in combination.

The combination agents are administered to the mammal in a therapeutically effective amount. The term "therapeutically effective amount" means the amount of the compounds of the present invention which, when administered alone or in combination with an additional therapeutic agent to a mammal, is effective in treating the desired disease/condition, eg, NASH.

Preferred agents for the treatment of non-alcoholic steatohepatitis (NASH) and/or non-alcoholic fatty liver disease (NAFLD) (i.e. anti-NASH and anti-NAFLD agents) are an acetyl-CoA carboxylase (ACC) inhibitor, a ketohexoginase (KHK) inhibitor, GLP-1 receptor agonist, FXR agonist, CB1 antagonist, ASK1 inhibitor, CCR2 and/or CCR5 inhibitor, PNPLA3 inhibitor, hydroxysteroid-17-β-dehydrogenase (HSD17B13) inhibitor, DGAT1 inhibitor, F21 analog, FGF19 analog, SGLT2 inhibitor, agonist PPAR, AMPK activator, SCD1 inhibitor, or MPO inhibitor. The traditionally cited patent application PCT/IB2017/057577 filed on 01/12/2017 refers to a GLP-1 receptor agonist. More preferred are an FXR agonist, an apoptosis signal-regulating kinase 1 (ASK1) inhibitor, a PPAR agonist, a GLP-1 receptor agonist, an SGLT inhibitor, an ACC inhibitor, and a KHK inhibitor.

Given the activity of the compounds of the present invention against NASH/NAFLD, they may be co-administered with other drugs for the treatment of non-alcoholic steatohepatitis (NASH) and/or non-alcoholic fatty liver disease (NAFLD) and related diseases/conditions, such as orlistat (Orlistat), thiazolidinedione derivatives (TZD) and other insulin sensitizing drugs, FGF21 analogs, metformin, omega-3 ethyl esters (e.g. Lovaza), fibrates, hydroxymethylglutaryl coenzyme A reductase inhibitors, ezetimibe, probucol, urodeoxycholic acid, TGR5 agonists, FXR agonists, vitamin E, betaine, pentoxifylline, CB1 antagonists, carnitine, N-acetylcysteine, reduced glutathione, lorcaserin, naltrexone-buproprion combination, SGLT2 inhibitors (including dapagliflozin, canagliflozin, empagliflozin, tofogliflozin, ertugliflozin, ASP -1941, THR1474, TS-071, ISIS388626 and LX4211 as well as SGLT2 inhibitors disclosed in WO2010023594) , phentermine, topiramate, GLP-1 receptor agonists, GIP receptor agonists, GLP-1/glucagon receptor dual agonists (i.e. OPK88003, MEDI0382, JNJ-64565111, NN9277, BI 456906), GLP-1/GIP receptor dual agonists (i.e. tirzepatide (LY3298176), NN9423), angiotensin receptor blockers, acetyl-CoA carboxylase (ACC) inhibitor, inhibitor BCKDK, ketohexokinase (KHK) inhibitor, ASK1 inhibitors, branched-chain alpha keto acid dehydrogenase kinase (BCBK) inhibitors, CCR2 and/or CCR5 inhibitors, PNPLA3 inhibitors, DGAT1 inhibitors, FGF21 analog, analogs FGF19, PPAR agonists, FXR agonists, AMPK activators, SCD1 inhibitors, or MPO inhibitors.

Typical examples of ACC inhibitors include 4-(4-[(1-isopropyl-7-oxo-1,4,6,7-tetrahydro-1'H-spiro[indazol-5,4'-piperidin]-1'-yl )carbonyl]-6-methoxypyridin-2-yl)benzoic acid and firsocostat (GS-0976) and their pharmaceutically acceptable salts.

Representative examples of DGAT2 inhibitors include (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide;

2-(5-((3-ethoxy-5-fluoropyridin-2-yl)oxy)pyridin-3-yl)-N-((3R,4S)-4-fluoropiperidin-3-yl)pyrimidine-5-carboxamide ;

2-(5-((3-ethoxy-5-fluoropyridin-2-yl)oxy)pyridin-3-yl)-N-((3S,5S)-5-fluoropiperidin-3-yl)pyrimidine-5-carboxamide ;

2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-((3R,4S)-4-fluoropiperidin-3-yl)pyrimidine-5-carboxamide;

2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-((3R,4R)-4-fluoropiperidin-3-yl)pyrimidine-5-carboxamide;

2-(5-((3-ethoxy-5-fluoropyridin-2-yl)oxy)pyridin-3-yl)-N-((3R,4R)-4-fluoropiperidin-3-yl)pyrimidine-5-carboxamide ; and

2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-((3S,5S)-5-fluoropiperidin-3-yl)pyrimidine-5-carboxamide.

Examples of suitable antidiabetic agents include, for example, insulins, metformin, DPPIV inhibitors, GLP-1 receptor agonists, analogs and mimetics, SGLT1 and SGLT2 inhibitors. Подходящие противодиабетические средства включают ингибиторы ацетил-КоА-карбоксилазы (АКК), например описанные в WO2009144554, WO2003072197, WO2009144555 и WO2008065508, ингибиторы диацилглицерол-O-ациaтрансферазы 1 (DGAT-1), например описанные в WO09016462 или WO2010086820, AZD7687 или LCQ908, ингибиторы monoacylglycerol-O-acyltransferase, phosphodiesterase-10 (PDE-10) inhibitor, AMPK activator, sulfonylurea (eg, acetohexamide, chlorpropamide, diabines, glibenclamide, glipizide, glyburide, glimepiride, gliclazide, glipentide, gliquidone, glisolamide, tolazamide, and tolbutamide), meglitinide, α-amylase inhibitor (eg, tendamistat, trestatin, and AL-3688), α-glucoside hydrolase inhibitors (eg, acarbose), α-glucosidase inhibitor (eg, adiposin, camiglibose, emiglitate, miglitol, voglibose, pradimycin-Q, and salbostatin ), PPARγ agonist (eg, balaglitazone, ciglitazone, darglitazone, englitazone, isaglitazone, pioglitazone, and rosiglitazone), PPAR α/γ agonist (eg, CLX-0940, GW-1536, GW- 1929, GW-2433, KRP-297, L-796449, LR-90, MK-0767 and SB-219994), biguanide (eg, metformin), glucagon-like peptide 1 modulator (GLP-1 ), such as an agonist (eg, exendin-3 and exendin-4), liraglutide, albaglutide, exenatide (Byetta®), albiglutide, lixisenatide, dulaglutide, semaglutide, NN-9924, TTP-054, protein tyrosine phosphatase-1B inhibitor (protein tyrosine phosphatase-1B - PTP-1B) (e.g. troduskvemin, hyrtiosal extract and compounds described in Zhang, S., et al., Drug Discovery Today, 12(9/10), 373-381 (2007 )), SIRT-1 activator (e.g., resveratrol, GSK2245840 or GSK184072), dipeptidyl peptidase-IV (DPP-IV) inhibitor (e.g., described in WO2005116014, sitagliptin, vildagliptin, alogliptin, dutogliptin, linagliptin and saxagliptin), insulin secretion stimulator, fatty acid oxidation inhibitor, A2 antagonist, c-Jun N-terminal kinase (JNK) inhibitor, glucokinase (GKa) activators, t such as the activators described in WO2010103437, WO2010103438, WO2010013161, WO2007122482, TTP-399, TTP-355, TTP-547, AZD1656, ARRY403, MK-0599, TAK-329, AZD5658, or GKM-00 inhibitor glycogen phosphorylase (e.g. GSK1362885), VPAC2t receptor agonist, SGLT2 inhibitors such as those described in E.C. Chao et al. Nature Reviews Drug Discovery 9, 551-559 (July 2010) including dapagliflozin, canagliflozin, empagliflozin, tofogliflozin (CSG452), ertugliflozin, ASP-1941, THR1474, TS-071, ISIS388626 and LX4211 and compounds disclosed in WO2010023594, modulator glucagon receptor, such as that described in Demong, D.E. et al. Annual Reports in Medicinal Chemistry 2008, 43, 119-137, GPR119 modulators, in particular agonists, such as those described in WO2010140092, WO2010128425, WO2010128414, WO2010106457, as well as Jones, R.M. et al., Medicinal Chemistry 2009, 44, 149-170 (e.g., MBX-2982, GSK1292263, APD597, and PSN821), FGF21 derivatives and analogs, such as those described in Kharitonenkov, A. et al., Current Opinion in Investigational Drugs 2009 , 10(4)359-364, modulators of the TGR5 receptor (also called GPBAR1), in particular agonists, such as those described in Zhong, M., Current Topics in Medicinal Chemistry, 2010, 10(4), 386-396, and INT777, GPR40 agonists, such as those described in Medina, J.C., Annual Reports in Medicinal Chemistry, 2008, 43, 75-85, including but not limited to TAK-875, GPR120 modulators, in particular agonists, high affinity nicotinic acid receptor activators (HM74A) and SGLT1 inhibitors such as GSK1614235. An additional representative list of antidiabetic agents that can be combined with the compounds of the present invention can be found, for example, in WO2011005611 (page 28 line 35 - page 30 line 19). Preferred antidiabetic agents are metformin and DPP-IV inhibitors (eg sitagliptin, vildagliptin, alogliptin, dutogliptin, linagliptin and saxagliptin). Other antidiabetic agents may include carnitine palmitoyltransferase enzyme inhibitors or modulators, fructose 1,6-diphosphatase inhibitors, aldose reductase inhibitors, mineralocorticoid receptor inhibitors, TORC2 inhibitors, CCR2 and/or CCR5 inhibitors, PKC isoform inhibitors (e.g. PKCα, PKCβ, PKCγ), fatty acid synthetase inhibitors, serine palmitoyltransferase inhibitors, GPR81, GPR39, GPR43, GPR41, GPR105, Kv1.3 modulators, retinol-binding protein, glucocorticoid receptor, somatostatin receptors (e.g. SSTR1, SSTR2, SSTR3 and SSTR5), PDHK2 or PDHK4 inhibitors or modulators, MAP4K4 inhibitors, IL1 family modulators including IL1beta, RXRalpha modulators. In addition, suitable antidiabetic agents include the mechanisms described in Carpino, P.A., Goodwin, B. Expert Opin. Ther. Pat, 2010, 20(12), 1627-51.

Suitable anti-obesity agents include 11β-hydroxy steroid dehydrogenase-1 (11β-hydroxy steroid dehydrogenase-1-11β-HSD type 1) inhibitors, stearoyl-CoA desaturase-1 (SCD-1) inhibitor, MCR agonists -4, cholecystokinin-A (cholecystokinin-A - CCK-A) agonists, monoamine reuptake inhibitors (such as sibutramine), sympathomimetics, β ₃ -adrenergic agonists, dopamine receptor agonists (such as bromocriptine), melanocyte-stimulating hormone analogs, agonists 5HT2c, melanin-concentrating hormone antagonists, leptin (OB protein), leptin analogs, leptin agonists, galanin antagonists, lipase inhibitors (such as tetrahydrolipstatin, i.e. orlistat), anorectics (such as bombesin agonists), neuropeptide Y (neuropeptide- Y - NPY) (for example, NPY Y5 antagonists), PYY _3-36 (including its analogs), thyromimetics, dehydrosterone or its analogs, glucocorticoid agonists or antagonists, arexin antagonists receptors, glucagon-like peptide-1 receptor agonists, ciliary neurotropic factors (such as Axokine™, available from Regeneron Pharmaceuticals, Inc., Tarrytown, NY and Procter & Gamble Company, Cincinnati, OH), agouti-related protein inhibitors - AGRP), ghrelin antagonists, histamine 3 antagonists or inverse agonists, neuromedin U agonists, MTP/ApoB inhibitors (e.g. gut-selective MTP inhibitors such as dirlotapide), opioid antagonist, orexin receptor antagonists, naltrexone-buproprion combination, etc. P.

Preferred anti-obesity agents for use in the combination of the present invention include gut-selective MTP inhibitors (e.g., dirlotapide, mitratapide, and implitapide, R56918 (CAS no. 403987) and CAS no. 913541-47-6), CCKa agonists (e.g., N-benzyl -2-[4-(1H-indol-3-ylmethyl)-5-oxo-1-phenyl-4,5-dihydro-2,3,6,10b-tetraazabenzo[e]azulen-6-yl]-N -isopropylacetamide described in PCT Publication No. WO 2005/116034 or US Publication No. 2005-0267100 A1), 5HT2c agonists (for example, lorcaserin), MCR4 agonists (for example, compounds described in US Patent No. 6818658), lipase inhibitor (for example, Cetilistat), PYY _3-36 (where "PYY _3-36 " when used herein includes its analogs such as pegylated PYY _3-36 such as those described in US Publication 2006/0178501), opioid antagonists (eg naltrexone ), naltrexone-buproprion combination, oleoyl-estrone (CAS # 180003-17-2), obinepitide (™30338), pramlintide (Symlin®), tesofensine (NS2330), leptin, liraglutide, bromocriptine, orlistat, exenatide (Byetta®), AOD-9604 (CAS No. 221231-10-3), phentermine, topiramate (trade name: Qsymia) and sibutramine. Preferably, the compounds of the present invention and the combination therapy are administered in conjunction with exercise and a sensible diet.

The compounds of the present invention can be used in combination with cholesterol-modulating agents (including cholesterol-lowering agents) such as a lipase inhibitor, an HMG-CoA reductase inhibitor, an HMG-CoA synthase inhibitor, an HMG-CoA reductase gene expression inhibitor, an HMG gene expression inhibitor -CoA synthase, MTP/Apo B secretion inhibitor, CETP inhibitor, bile acid absorption inhibitor, cholesterol absorption inhibitor, cholesterol synthesis inhibitor, squalene synthetase inhibitor, squalene epoxidase inhibitor, squalene cyclase inhibitor, combined squalene epoxidase/squalene cyclase inhibitor, fibrate, niacin, ion exchange resin , an antioxidant, an ACAT inhibitor or a bile acid sequestrant, or an agent such as mipomersen.

Examples of suitable cholesterol/lipid-lowering agents and lipid profile therapeutics include HMG-CoA reductase inhibitors (eg, pravastatin, pitavastatin, lovastatin, atorvastatin, simvastatin, fluvastatin, NK-104 (also known as itavastatin, nisvastatin, or nisbastatin) and ZD- 4522 (also known as rosuvastatin, atavastatin, or visastatin); squalene synthetase inhibitors; fibrates (eg, fibrozil, fenofibrate, clofibrate); bile acid sequestrants (such as questran, colestipol, colesevelam); ACAT inhibitors; MTP inhibitors; lipoxygenase inhibitors; absorption inhibitors cholesterol (eg, ezetimibe); nicotinic acid pharmaceuticals (eg, niacin, niacor, slo-niacin); omega-3 fatty acids; and cholesterol ester transport protein inhibitors. Other anti-atherosclerotic agents include PCSK9 modulators (eg, alirocumab and evolocumab) .

In another embodiment of the present invention, the compounds of the present invention may be co-administered with pharmaceutical agents for the treatment of non-alcoholic steatohepatitis (NASH) and/or non-alcoholic fatty liver disease (NAFLD), such as orlistat, TZD and other insulin sensitizing agents, FGF21 analogs, metformin , omega-3 esters (eg, lovase), fibrates, HMG-CoA reductase inhibitors, ezitimibe, probucol, urodeoxycholic acid, TGR5 agonists, FXR agonists, vitamin E, betaine, pentoxifylline, CB1 antagonists, carnitine, N- acetylcysteine, reduced glutathione, lorcaserin, naltrexone-buproprion combination, SGLT2 inhibitors, phentermine, topiramate, incretin analogs (GLP and GIP), and angiotensin receptor blockers.

In another embodiment of the present invention, the additional pharmaceutical agent is selected from the group consisting of cysteamine or a pharmaceutically acceptable salt thereof, cystamine or a pharmaceutically acceptable salt thereof, an antioxidant, lecithin, a vitamin B complex, bile salt preparations, cannabinoid 1 (CB1) receptor antagonists, reverse cannabinoid 1 (CB1) receptor agonists, regulators of the activity of peroxisome proliferator-activated receptors, a benzothiazepine or benzothiepine derivative, an antisense RNA construct for inhibition of protein tyrosine phosphatase PTPRU, piperidine linked through a heteroatom and its derivatives, an azacyclopentane derivative capable of inhibiting stearoyl-coenzyme alpha- delta-9-desaturase, an acylamide compound having the activity of a secretion stimulant or an inducer of adiponectin activity, a quaternary ammonium compound, glatiramer acetate, pentatraxine proteins, an HMG-CoA reductase inhibitor, n-acetylcysteine, co an isoflavone, a macrolide antibiotic, a galectin inhibitor, an antibody, or any combination thereof.

Additional therapeutic agents include anticoagulants or agents that inhibit coagulation, antiplatelet or platelet inhibitors, thrombin inhibitors, thrombolytic or fibrinolytic agents, antiarrhythmic agents, antihypertensive agents, calcium channel blockers (L-type and T-type), cardiac glycosides, diuretics , mineralocorticoid receptor agonists, NO-providing agents such as organic nitrates, NO-promoting agents such as phosphodiesterase inhibitors, cholesterol/lipid-lowering agents, lipid profile therapeutic agents, antidiabetic agents, antidepressants, anti-inflammatory agents (steroidal and non-steroidal), anti-osteoporosis agents, hormone replacement therapeutic agents, oral contraceptives, anti-obesity agents, anxiolytics, anti-proliferative agents, anti-tumor agents, anti-ulcer agents and anti-gastroesophageal agents phageal reflux disease, growth hormone and/or growth hormone secretagogues, thyroid mimetics (including thyroid hormone receptor antagonists), anti-infective agents, antiviral agents, bactericidal and fungicidal agents.

Critical care agents include, for example, dobutamine, dopamine, epinephrine, nitroglycerin, nitroprusside, and the like.

Combination agents suitable for the treatment of vasculitis include, for example, azathioprine, cyclophosphamide, mycophenolate, mofetil, rituximab, and the like.

In another embodiment, the present invention relates to a combination wherein the third pharmaceutical agent is at least one pharmaceutical agent selected from a factor Xa inhibitor, an anticoagulant agent, an antiplatelet agent, a thrombin inhibitor agent, a thrombolytic agent, and a fibrinolytic agent. Representative examples of factor Xa inhibitors include apixaban and rivaroxaban. Examples of suitable anticoagulants for use in combination with the compounds of the present invention include heparins (eg unfractionated and low molecular weight heparins such as enoxaparin and dalteparin).

In another preferred embodiment, the third pharmaceutical agent is at least one selected from warfarin, dabigatran, unfractionated heparin, low molecular weight heparin, synthetic pentasaccharide, hirudin, argatroban, aspirin, ibuprofen, naproxen, sulindac, indomethacin, mefenamate, droxicam, diclofenac , sulfinpyrazone, piroxicam, ticlopidine, clopidogrel, tirofiban, eptifibatide, abciximab, melagatran, disulfatohirudin, tissue plasminogen activator, modified tissue plasminogen activator, anistreplase, urokinase and streptokinase.

A preferred third pharmaceutical agent is at least one antiplatelet agent. Particularly preferred antiplatelet agents are aspirin and clopidogrel.

The term "antiplatelet agents" (or "platelet-inhibiting agents"), as used herein, means agents that inhibit platelet function, eg, inhibition of platelet aggregation, adhesion, or granular secretion. These agents include, but are not limited to, various known non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, naproxen, sulindac, indomethacin, mefenamate, droxicam, diclofenac, sulfinpyrazone, piroxicam and their pharmaceutically acceptable salts and prodrugs. Among NSAIDS, aspirin (acetylsalicylic acid or ASA) and COX-2 inhibitors such as Celebrex or Piroxicam are preferred. Other suitable platelet inhibitors include IIb/IIIa antagonists (eg, tirofiban, eptifibatide, and abciximab), thromboxane-A2 receptor antagonists (eg, ifetroban), thromboxane A2 synthetase inhibitors, PDE-III inhibitors (eg, pletal, dipyridamole), and their pharmaceutically acceptable salts and prodrugs.

The term "antiplatelet agents" (or "platelet-inhibiting agents"), when used herein, is intended to include ADP (adenosine diphosphate) receptor antagonists, preferably P2Y1 and P2Y12 purinergic receptor antagonists, more preferably P2Y12. Preferred P2Y12 receptor antagonists include ticagrelor, prasugrel, ticlopidine and clopidogrel and their pharmaceutically acceptable salts and prodrugs. Clopidogrel is even more preferred. Ticlopidine and clopidogrel are also preferred compounds because they are known to be mild to the gastrointestinal tract when applied.

The term "thrombin inhibitors" (or "antithrombin agents"), when used in the present invention, means inhibitors of serine prothiasis thrombin. Thrombin inhibition interferes with various thrombin-mediated processes, such as thrombin-mediated platelet activation (i.e., for example, platelet aggregation and/or granular secretion of plasminogen activator inhibitor-1 and/or serotonin) and/or fibrin formation. Thrombin inhibitors are known to those skilled in the art and are intended to be used in combination with the compounds of the present invention. Such inhibitors include, but are not limited to, boroarginine derivatives, boropeptides, dabigatran, heparins, hirudin, argatroban, and melagatran, including their pharmaceutically acceptable salts and prodrugs. Boroarginine derivatives and boropeptides include N-acetyl and peptide boronic acid derivatives such as C-terminal alpha-aminoboronic acid derivatives of lysine, ornithine, arginine, homoarginine and their corresponding isothiouronium analogs. The term "hirudin" as used herein includes suitable derivatives or analogs of hirudin referred to herein by hirulologists, such as disulfatohirudin. The term "thrombolytics" or "fibrinolytics" (or "thrombolytics" or "fibrinolytics"), when used in the present description, means pharmaceutical agents that lyse blood clots (thrombi). Such agents include tissue plasminogen activator (natural or recombinant) and its modified forms, anistreplase, urokinase, streptokinase, tenecteplase (TNK), lanoteplase (nPA), factor VIIa inhibitors, PAI-1 inhibitors (i.e. inactivators of tissue plasminogen activator inhibitors ), alpha-2-antiplasmin inhibitors, and anisoylated plasminogen activator-streptokinase complex, including pharmaceutically acceptable salts and prodrugs thereof. The term "anistreplase" as used herein refers to an anisoylated plasminogen activator-streptokinase complex as described, for example, in EP 028489, the contents of which are incorporated herein by reference. The term "urokinase", when used in the present description, means both double-chain and single-chain urokinase, the latter also referred to in the present description as prourokinase.

Examples of suitable antiarrhythmic agents include Class I pharmaceuticals (such as propafenone); class II pharmaceuticals (such as metoprolol, atenolol, carvedilol and propranolol); class III pharmaceuticals (such as sotalol, dofetilide, amiodarone, azimilide and ibutilide); class IV pharmaceuticals (such as diltiazem and verapamil); potassium (K+) channel activators such as I _Ach inhibitors and I _Kur inhibitors (eg as described in WO01/40231).

Compounds of the present invention can be co-administered with anti-heart failure agents such as ACE inhibitors (eg captopril, enalapril, fosinopril, lisinopril, perindopril, quinapril, ramipril, trandolapril), angiotensin II receptor blockers (eg candesartan, losartan, valsartan) , angiotensin receptor inhibitors naprilysin (sacubitril/valsartan), I _f channel blocker ivabradine, beta-adrenergic blockers (eg bisoprolol, metoprolol succinate, carvedilol), aldesterone antagonist (eg spironolactone, eplerenone), hydralazine and isosorbide dinitrate, diuretics ( e.g. furosemide, bumetanide, torsemide, chlorothiazide, amiloride, hydrochlorothiazide, indapamide, metolazone, triamterene) or digoxin.

The compounds of the present invention may be used in combination with antihypertensive agents and such antihypertensive activity is readily determined by those skilled in the art according to standard assays (eg blood pressure measurement). Examples of suitable antihypertensive agents include alpha-blockers; beta-blockers; calcium channel blockers (eg diltiazem, verapamil, nifedipine and amlodipine); vasodilators (eg, hydralazine), diuretics (eg, chlorothiazide, hydrochlorothiazide, flumethiazide, hydroflumethiazide, bendroflumethiazide, methylchlorothiazide, trichloromethiazide, polythiazide, benzthiazide, ethacrynic acid, tricrinafen, chlorthalidone, torasemide, furosemide, indapamide, metozolone, musolominone, bumetanide, triamterene, amiloride, spironolator); renin inhibitors; ACE inhibitors (eg captopril, zofenopril, fosinopril, enalapril, keranopril, cilazopril, delapril, pentopril, perindopril, quinapril, ramipril, trandolapril, lisinopril); AT-1 receptor antagonists (eg losartan, irbesartan, valsartan); angiotensin/neprilysin receptor inhibitors (sacubitril/valsartan); beta-adrenergic receptor blockers (eg bisoprolol, metaprolol succinate, carvedilol); ET receptor antagonists (eg, sitaxentan, anthrasentan, and compounds described in US Pat. Nos. 5,612,359 and 6,043,265); dual ET/AII antagonists (eg the compounds described in WO 00/01389); inhibitors of neutral endopeptidase (neutral endopeptidase - NEP); vasopeptidase inhibitors (dual NEP-ACE inhibitors) (eg, hemopatrilat and nitrates). A typical antianginal agent is ivabradine.

Examples of suitable calcium channel blockers (L-type or T-type) include diltiazem, varapamil, nifedipine, amlodipine and mibefradil.

Examples of suitable cardiac glycosides include digitalis and ouabain.

In one embodiment of the present invention, the compounds of the present invention may be co-administered with one or more diuretics. Examples of suitable diuretics include (a) loop diuretics such as furosemide (eg LASIX™), torsemide (eg DEMADEX™), bemethanide (eg BUMEX™) and ethacrynic acid (eg EDECRIN™); (b) thiazide diuretics such as chlorothiazide (eg DIURIL™, ESIDRIX™ or HYDRODIURIL™), hydrochlorothiazide (eg MICROZIDE™ or ORETIC™), benzthiazide, hydroflumethiazide (eg SALURON™), bendroflumethiazide, methychlorothiazide, polythiazide, trichlormethiazide and indapamide (eg LOZOL™); (c) phthalimidine diuretics such as chlorthialidon (eg HYGROTON™) and metolazone (eg ZAROXOLYN™); (d) quinazoline diuretics such as quinerazone; and (e) potassium-sparing diuretics such as triamterene (eg DYRENIUM™) and amiloride (eg MIDAMOR™ or MODURETIC™).

In another embodiment of the present invention, the compounds of the present invention may be co-administered with a loop diuretic. In yet another embodiment, the loop diuretic is selected from furosemide and torsemide. In yet another embodiment, the compounds of the present invention may be co-administered with furosemide. In yet another embodiment, the compounds of the present invention may be co-administered with torsemide, which may optionally be in a controlled or modified release form.

In another embodiment of the present invention, the compounds of the present invention may be co-administered with a thiazide diuretic. In another embodiment, the thiazide diuretic is selected from the group consisting of chlorothiazide and hydrochlorothiazide. In yet another embodiment, the compounds of the present invention may be co-administered with chlorothiazide. In yet another embodiment, the compounds of the present invention may be co-administered with hydrochlorothiazide.

In another embodiment of the present invention, the compounds of the present invention may be co-administered with a phthalimidine diuretic. In yet another embodiment, the phthalimidine diuretic is chlorthalidone. Examples of suitable mineralocorticoid receptor antagonists include spironolactone and eplerenone. Examples of suitable phosphodiesterase inhibitors include PDE II inhibitors (such as cilostazol); and PDE V inhibitors (such as sildenafil).

Those skilled in the art will appreciate that the compounds of the present invention may be used in conjunction with other cardiovascular or cerebrovascular treatments, including PCI, stenting, drug-eluting stents, stem cell therapy, and medical devices such as implantable pacemakers, defibrillators, or cardiac resynchronization therapy.

In particular, when used as a dosage unit form, there is the possibility of chemical interaction between the combined active ingredients. For this reason, when the first therapeutic agent and the second therapeutic agent are combined into the same unit dose dosage form, the formulation is prepared such that although the active ingredients are combined into the unit dose dosage form, physical contact between the active ingredients is minimized (then reduced). For example, one active ingredient may be enteric coated. By enteric coating of one of the active ingredients, not only can contact between the combined active ingredients be minimized, but also the release of one of these components in the gastrointestinal tract can be controlled so that one of these components is not released in the stomach, but in the intestine. One of the active ingredients may also be coated with a material that releases the active ingredient in a sustained manner throughout the gastrointestinal tract and also serves to minimize physical contact between the combined active ingredients. In addition, the sustained release component can be additionally coated with an enteric coating so that the release of this component occurs only in the intestine. Yet another approach is to provide a combination product in which one component is coated with a sustained and/or enteric release polymer and the other component is also coated with a polymer such as low viscosity hydroxypropyl methylcellulose (HPMC) or other suitable materials that are known in the art, for additional separation of active components. The polymer coating serves to form an additional barrier that prevents interaction with another component.

These and other methods of minimizing contact between the components of the combination products of the present invention, administered in the same dosage form or administered in separate forms, but at the same time by the same route of administration, will be understood by those skilled in the art after reading this description.

In combination therapy treatment, the compounds of the present invention and other drugs are administered to mammals (eg, humans, males or females) by standard methods.

The dose of each therapeutic agent, such as Compound A, Compound D, and any additional therapeutic agent, will generally depend on a number of factors including the health status of the subject being treated, the amount of treatment required, the nature and type of concomitant treatment, if any, and the frequency of treatment administered. and the nature of the desired effect. Typically, the dose of each therapeutic agent is in the range of from about 0.001 mg to about 100 mg per kilogram of individual body weight per day, preferably from about 0.1 mg to about 10 mg per kilogram of individual body weight per day. However, some variation in the overall dosage range may also be required depending on the age and body weight of the subject being treated, the intended route of administration, the specific anti-obesity agent being administered, and the like. Determination of dosage ranges and optimal dosages for a particular patient is also within the skill of the art, having the benefit of the present invention.

In accordance with the methods of treatment of the present invention, a compound of the present invention, or combinations of a compound of the present invention and at least one additional pharmaceutical agent (referred to in the description as "combination"), is administered to a subject in need of such treatment, preferably in the form of a pharmaceutical composition. In the combination aspect of the present invention, the compound of the present invention and at least one other pharmaceutical agent (eg, another anti-obesity agent) may be administered separately or in a pharmaceutical composition comprising both active ingredients. Typically, such administration is preferably oral.

When a combination of a compound of the present invention and at least one other pharmaceutical agent is administered together, such administration may be sequential in time or simultaneous. Simultaneous administration of drug combinations is preferred. When administered sequentially, the compound of the present invention and the additional pharmaceutical agent may be administered in any order. Typically, such administration is preferably oral. Especially preferred is oral and simultaneous administration. When the compound of the present invention and the additional pharmaceutical agent are administered sequentially, they may be administered in the same or different ways.

In accordance with the methods of the present invention, the compound of the present invention or combination is preferably administered in the form of a pharmaceutical composition. Accordingly, a compound of the present invention or combination may be administered to a patient alone or together in any conventional dosage form for oral, rectal, transdermal, parenteral (e.g., intravenous, intramuscular, or subcutaneous), intracisternal, intravaginal, intraperitoneal, topical (e.g., in powder form). , ointment, cream, spray or lotion), buccal or nasal administration (for example, spray, drops or inhalation).

The compounds of the present invention or combinations may be administered alone, but will generally be administered in admixture with one or more suitable pharmaceutical excipients, adjuvants, diluents or carriers known in the art and selected in consideration of the intended route of administration and standard pharmaceutical practice. The compound of the present invention, or combination, may be formulated to provide immediate, delayed, modified, sustained, pulsed, or controlled release dosage forms, depending on the desired route of administration and the specificity of the release profile commensurate with therapeutic needs.

The pharmaceutical composition comprises a compound of the present invention or combination in an amount typically in the range of from about 1% to about 75%, 80%, 85%, 90% or even 95% (by weight) of the composition, usually in the range from about 1%, 2 % or 3% to about 50%, 60% or 70%, more often in the range from about 1%, 2% or 3% to less than 50%, for example about 25%, 30% or 35%.

Methods for preparing various pharmaceutical compositions with a specific amount of active compound are known to those skilled in the art (see, for example, Remington: The Practice of Pharmacy, Lippincott Williams and Wilkins, Baltimore Md. 20.sup.th ed. 2000).

Compositions suitable for parenteral injection generally include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution to obtain sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers or diluents (including solvents and drug carrier vehicles) include water, ethanol, polyhydric alcohols (propylene glycol, polyethylene glycol, glycerin, and the like), suitable mixtures thereof, triglycerides, including vegetable oils such as olive oil, and edible organic esters such as ethyl oleate. A preferred carrier is Miglyol® (trademark) a propylene glycol ester of caprylic/capric acid (e.g. Miglyol.R™.812, Miglyol.R™.829, Miglyol.R™.840), available from Condea Vista Co., Cranford, N.J. The necessary fluidity can be maintained, for example, by applying a coating such as lecithin, providing the required particle size in the case of a dispersion, and using surfactants.

These compositions for parenteral injection may also contain excipients such as preservatives, wetting agents, emulsifiers and dispersants. Prevention of microbial contamination of the compositions can be accomplished by using various bactericidal and fungicidal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may be desirable to use isotonic agents, eg sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical compositions can be achieved by the use of additives capable of delaying absorption, such as aluminum monostearate and gelatin.

Solid dosage forms for oral administration include capsules, tablets, chewable tablets, lozenges, pills, powders, and multiparticulate preparations (granules). In such solid dosage forms, the compound of the present invention or combination is mixed with at least one inert excipient, diluent or carrier. Suitable excipients, diluents or carriers include materials such as sodium citrate or dicalcium phosphate and/or (a) one or more fillers or diluents (e.g. microcrystalline cellulose (available as Avicel.™. from FMC Corp.), starches, lactose, sucrose , mannitol, silicic acid, xylitol, sorbitol, dextrose, calcium hydrogen phosphate, dextrin, alpha-cyclodextrin, beta-cyclodextrin, polyethylene glycol, medium chain fatty acids, titanium oxide, magnesium oxide, alumina, etc.); (b) one or more binders (for example, carboxymethylcellulose, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, gelatin, gum arabic, ethylcellulose, polyvinyl alcohol, pullulan, pregelatinized starch, agar, tragacanth, alginates, gelatin, polyvinylpyrrolidone, sucrose, gum arabic, etc. .); (c) one or more humectants (eg, glycerin, etc.); (d) one or more disintegrants (eg, agar-agar, calcium carbonate, potato or tapioca starch, allginic acid, certain complex silicates, sodium carbonate, sodium lauryl sulfate, sodium starch glycolate (available as Explotab™ from Edward Mendell Co.) , cross-linked polyvinylpyrrolidone, A-type croscarmellose sodium (available as Ac-di-sol.™.), polyacrylin potassium (polyacrylin potassium) (ion exchange resin) and the like); (e) one or more dissolution retarders (eg, paraffin, etc.); (f) one or more absorption accelerators (eg, quaternary ammonium compounds, etc.); (g) one or more wetting agents (eg, cetyl alcohol, glycerol monostearate, etc.); (h) one or more adsorbents (eg kaolin, bentonite, etc.); and/or (i) one or more lubricants (e.g., talc, calcium stearate, magnesium stearate, stearic acid, polyoxyl stearate, cetanol, talc, hydrogenated castor oil, sucrose fatty acid esters, dimethylpolysiloxane, microcrystalline wax, yellow beeswax , white beeswax, solid polyethylene glycols, sodium lauryl sulfate, etc.). In the case of capsules and tablets, dosage forms may also include buffering agents.

Solid compositions of a similar type can also be used as fillers in soft or hard gelatin capsules using excipients such as lactose, milk sugar, as well as high molecular weight polyethylene glycols, and the like.

Solid dosage forms such as tablets, dragees, capsules and granules can be prepared with coatings and shells such as enteric coatings and others well known in the art. They may also contain opacifying agents and may be presented in compositions that release the compound of the present invention and/or the additional pharmaceutical agent in a delayed manner. Examples of embedding compositions that can be used are polymeric materials and waxes. The drug may also be presented in microencapsulated form, if appropriate, with one or more of the above excipients.

In tablets, the active ingredient will typically comprise less than 50% (by weight) of the formulation, such as less than about 10%, such as 5% or 2.5% by weight. The bulk of such a preparation is made up of excipients, diluents, disintegrants, lubricants, and possibly flavors. The composition of these excipients is well known in the art. Often fillers/diluents contain mixtures of two or more of the following: microcrystalline cellulose, mannitol, lactose (all types), starch and dicalcium phosphate. Filler/diluent mixtures typically comprise less than 98% of the formulation, preferably less than 95%, eg 93.5%. Preferred disintegrants include Ac-di-sol.™., Explotab.™., starch and sodium lauryl sulfate. The disintegrating agent, when present, typically makes up less than 10% of the formulation, or less than 5%, such as about 3%. The preferred lubricant is magnesium stearate. Lubricant, when present, typically makes up less than 5% of the formulation, or less than 3%, such as about 1%.

Tablets can be produced by standard tableting processes, such as direct compression or wet or dry granulation or melt granulation and extrusion. Tablet cores may be single or multilayer and may be coated with suitable coatings known in the art.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the compound of the present invention or combination, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol , 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil, etc.), Miglyole™ (available from CONDEA Vista Co., Cranford, N.J.) , glycerin, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters or mixtures of these substances, and the like.

In addition to such inert diluents, the composition may also include excipients such as wetting agents, emulsifying and suspending agents, sweeteners, flavors and flavors.

Oral liquid forms of the compounds of the present invention, or combinations thereof, include solutions in which the active compound is completely dissolved. Examples of solvents include all known pharmaceutical solvents suitable for oral administration, in particular solvents in which the compounds of the invention exhibit good solubility, such as polyethylene glycol, polypropylene glycol, edible oils, and systems based on glycerol and glycerides. Glycerol and glyceride systems may include, for example, the following branded products (and related generics): Captex™ 355EP (glyceryl tricaprylate/caprate, from Abitec, Columbus Ohio), Crodamol™ GTC/C (medium chain triglyceride, from Croda, Cowick Hall , UK) or Labrafac™ CC (medium chain triglyceride, from Gattefosse), Captex™ 500P (glyceryl triacetate, i.e. triacetin, from Abitec), Capmul™ MCM (medium chain mono- and diglycerides, from Abitec), Migyol™ 812 (caprylic /capric triglyceride, from Condea, Cranford N.J.), Migyol™ 829 (caprylic/capric/succinic triglyceride, from Condea), Migyol™ 840 (propylene glycol dicaprylate/dicaprate, from Condea), Labrafil™ M1944CS (oleoyl macrogol-6-glycerides, from Gattefosse ), Peceol™ (glyceryl monooleate, from Gattefosse) and Maisine™ 35-1 (glyceryl monooleate, from Gattefosse). Of particular interest are medium chain (about C8 to C10) triglyceride oils. These solvents often make up the majority of the composition, ie. greater than about 50%, typically greater than about 80%, such as about 95% or 99%. Adjuvants and additives can also be included in solvent formulations, mainly as taste masking agents, flavor improvers and additives, antioxidants, stabilizers, texture and viscosity modifiers, and solubilizers.

Suspensions, in addition to the compound of the present invention or combination, may additionally contain carriers such as suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitol esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, etc. P.

Compositions for rectal or vaginal administration preferably include suppositories which can be prepared by admixing a compound of the present invention or combination with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ordinary room temperature, but liquid at body temperature and therefore melt in the rectum or vaginal cavity, thus releasing the active ingredient(s).

Dosage forms for topical administration of the compounds of the present invention or combinations include ointments, creams, lotions, powders and sprays. The drugs are mixed with a pharmaceutically acceptable excipient, diluent or carrier, as well as any preservatives, buffering agents or propellants that may be required.

If the compounds are poorly soluble in water, for example the solubility is less than 1 μg/ml, then the preferred dosage form for these compounds are liquid formulations in solubilizing non-aqueous solvents such as the medium chain triglyceride oils discussed above.

Solid amorphous dispersions, including dispersions obtained in a spray drying process, are also the preferred dosage form for the poorly soluble compounds of the present invention. The term "solid amorphous dispersion" means a solid material in which at least a portion of the poorly soluble compound is in amorphous form and dispersed in a water-soluble polymer. The term "amorphous" means that the poorly soluble compound is not crystalline. The term "crystalline" means that the compound exhibits long-range order in three dimensions of at least 100 repeat units in each dimension. Thus, the term "amorphous" is intended to include not only material that is essentially without order, but also material that may have some small degree of order, but the order is in less than three dimensions and/or only at short distances. The amorphous material may be characterized using methods known in the art such as X-ray powder diffraction crystallography (XRD), solid NMR, or thermal methods such as differential scanning calorimetry (DSC).

Preferably, at least a major portion (ie, at least about 60% by weight) of the poorly soluble compound in the solid amorphous dispersion is amorphous. The compound may exist within a solid amorphous dispersion in relatively pure amorphous domains or regions, as a solid solution of the compound homogeneously distributed throughout the polymer, or in any combination of these states or states that are intermediate between them. Preferably, the solid amorphous dispersion is substantially homogeneous so that the amorphous compound is dispersed as homogeneously as possible throughout the polymer. The term "substantially homogeneous" when used herein means that the proportion of a compound that is present in relatively pure amorphous domains or regions within a solid amorphous dispersion is relatively small, less than 20 wt%, preferably less than 10 wt%, the total amount of the drug.

Water-soluble polymers suitable for use in solid amorphous dispersions must be inert, i.e. not chemically react with a poorly soluble compound in an unfavorable manner, be pharmaceutically acceptable, and have at least some solubility in aqueous solution at physiologically relevant pH (e.g., 1-8). The polymer may be neutral or ionizable, and its solubility in water should be at least 0.1 mg/ml at least at pH values in the range of 1-8.

Water-soluble polymers suitable for use in the present invention may be cellulosic or non-cellulosic. The polymers may be neutral or ionizable in aqueous solution. Of these, ionizable and cellulosic polymers are preferred, with ionizable cellulosic polymers being more preferred.

Typical water-soluble polymers include hydroxypropyl methyl cellulose acetate succinate (HPMCAS), hydroxypropyl methyl cellulose (HPMC), hydroxypropyl methyl cellulose phthalate (HPMCP), carboxymethyl ethyl cellulose (CMEC), phthalate cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), polyvinylpyrrolidone (polyvinylpyrrolidone - PVP), hydroxypropyl cellulose (hydroxypropyl cellulose - HPC), methyl cellulose (methyl cellulose - MC), block copolymers of ethylene oxide and propylene oxide (PEO/PPO, also known as poloxamers) and mixtures thereof. Particularly preferred polymers are HPMCA, HPMC, HPMCP, CMEC, CAP, CAT, PVP, poloxamers and mixtures thereof. Most preferred are HPMCAs (see European Patent Application Publication No. 0901786 A2, the contents of which are incorporated herein by reference).

Solid amorphous dispersions can be obtained in accordance with any method for obtaining solid amorphous dispersions, as a result of which at least a large part (at least 60%) of the poorly soluble compound is in the amorphous state. Such methods include mechanical and thermal methods, as well as methods using solvents. Typical examples of mechanical methods include milling and extrusion; thermal methods include methods for producing a melt, including high-temperature melting, solvent-modified melting, methods using melt solidification; and solvent methods include non-solvent deposition, spray coating, and spray drying (see, for example, the following US Pat. using grinding; and No. 5707646 and 4894235, which describe the preparation of dispersions using melt solidification; the content of all of these patents is incorporated into this description by reference). In a preferred process, a solid amorphous dispersion is obtained by spray drying as described in European Patent Application Publication No. 0901786 A2. In this method, the compound and the polymer are dissolved in a solvent such as acetone or methanol, and then the solvent is quickly removed from the solution by spray drying to form a solid amorphous dispersion. Solid amorphous dispersions can be obtained so that, if desired, they contain up to 99% of the mass. compounds, for example 1% wt., 5% wt., 10% wt., 25% wt., 50% wt., 75% wt., 95% wt. or 98% of the mass.

The solid dispersion can be used on its own as a dosage form or as a manufacturing-use-product (MUP) in the preparation of other dosage forms such as capsules, tablets, solutions or suspensions. An example of an aqueous suspension is a 1:1 (w/w) compound/HPMCASHF aqueous suspension spray-dried with a dispersion containing 2.5 mg/mL of the compound in 2% polysorbate-80. Solid dispersions for use in a tablet or capsule are usually mixed with other excipients or adjuvants conventionally used in such dosage forms. For example, a typical capsule filler contains a 2:1 (w/w) mixture of HPMCAS-MF compound/dispersion spray dried (60%), lactose (high flow) (15%), microcrystalline cellulose (e.g. Avicel(R0- 102) (15.8%), sodium starch (7%), sodium lauryl sulfate (2%) and magnesium stearate (1%).

HPMCAS resins are available as low, medium and high grade products such as Aqoa ^(R)-LF , Aqoat ^(R)-MF and Aqoat ^(R)-HF , respectively, from Shin-Etsu Chemical Co., LTD, Tokyo, Japan. In general, the higher grade products, MF and HF, are preferred.

The following describes exemplary formulations, dosages, etc. applicable to non-human animals. Administration of Compound A or a pharmaceutically acceptable salt thereof in combination with Compound D or a pharmaceutically acceptable salt thereof as two pharmaceutical agents or in combination with another pharmaceutical agent may be orally or non-orally.

An amount of Compound A or a pharmaceutically acceptable salt thereof with Compound D or a pharmaceutically acceptable salt thereof, together or in combination with another pharmaceutical agent, is administered such that an effective dose is obtained. Typically, the daily dose that is orally administered to an animal is from 0.01 to 1000 mg/kg of body weight, for example, from 0.01 to 300 mg/kg, from 0.01 to 100 mg/kg, from 0.01 to 50 mg/kg body weight, 0.01 to 25 mg/kg, 0.01 to 10 mg/kg, or 0.01 to 5 mg/kg. The daily dose of Compound A administered may be 2 mg, 3 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg or 50 mg. The daily dose can be divided into several doses, for example administered twice a day with a 12-hour interval of administration. For example, in some cases, the daily dose of Compound A may be administered at 15 mg every 12 hours. The daily dose of Compound D administered may be 50 mg, 100 mg, 200 mg or 300 mg. The daily dose can be divided into several doses, for example administered twice a day with an interval of 12 hours. For example, in some cases, the daily dose of Compound D may be administered by administering 300 mg every 12 hours.

Typically, a compound of the present invention (or combination) may be administered in drinking water such that a therapeutic dose of the compound is taken with daily water intake. The compound can be directly dosed into drinking water, preferably as a liquid water-soluble concentrate (eg, an aqueous solution of a water-soluble salt).

Typically, the compound of the present invention (or combination) may also be added directly to the feed or as an animal feed supplement, also referred to as a premix or concentrate. A premix or concentrate of a compound in an excipient, diluent or carrier is most commonly used to incorporate a pharmaceutical agent into a feed. Suitable excipients, diluents or carriers are liquid or solid, as desired, such as water, flours such as alfalfa meal, soybean meal, cotton meal, flax meal, corn cob meal and corn meal, molasses, urea, bone meal, and mineral mixtures that are commonly used in poultry feed. A particularly effective excipient, diluent or carrier is the animal feed itself, ie a small part of such feed. The carrier contributes to the uniform distribution of the mixture in the finished feed, with which the premix is mixed. Preferably, the mixture is thoroughly mixed into the premix and then into the feed. To this end, the compound may be dispersed or dissolved in a suitable oily carrier such as soybean oil, corn oil, cottonseed oil, etc., or in a volatile organic solvent, and then mixed with the carrier. It should be appreciated that the proportions of the compound in the concentrate can vary considerably, as the amount of the compound in the finished feed can be adjusted by mixing the premix in the feed in the appropriate proportion to obtain the desired compound content.

High potency concentrates can be blended by the feed manufacturer with a protein carrier such as soybean meal and other meals as described above to provide concentrated supplements that are suitable for direct animal feeding. In such cases, the animals can accept a normal diet. Alternatively, such concentrated supplements may be added directly to the feed to provide a nutritionally balanced finished feed containing a therapeutically effective level of a compound of the invention. The mixtures are thoroughly mixed by standard methods, such as a twin screw blender, to ensure uniformity.

If the additive is introduced as a top impregnation in the feed, it also promotes uniform distribution of the compound over the top of the impregnated feed.

Drinking water and feed used to increase lean meat and to improve the lean meat to fat ratio is typically prepared by mixing a compound of the present invention with enough animal feed to provide about 10 ^-3 to about 500 ppm. compounds in feed or water.

Preferred medicated feed for pigs, cattle, sheep and goats typically contains 1 to 400 grams of a compound of the present invention (or combination) per ton of feed, with the optimal amount for these animals typically being 50 to 300 grams per ton of feed.

Preferred poultry and pet foods typically contain 1 to 400 grams, preferably 10 to 400 grams, of a compound of the present invention (or combination) per ton of feed.

Typically, for parenteral administration to animals, the compounds of the present invention (or combinations thereof) may be formulated as a paste or granules and administered as an implant under the scalp or ear of an animal requiring increased lean buildup and improved lean meat to fat ratio.

Paste formulations can be prepared by dispersing the drug in a pharmaceutically acceptable oil such as peanut oil, sesame oil, corn oil, or the like.

Pellets containing a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof in combination with Compound D or a pharmaceutically acceptable salt thereof, the pharmaceutical composition or combination can be prepared by mixing Compound A or a pharmaceutically acceptable salt thereof with Compound D or a pharmaceutically acceptable salt thereof and a diluent such as carbovax, carnuba wax and the like, and a lubricant such as magnesium or calcium stearate may be added to improve the granulation process.

Of course, it is understood that more than one pellet may be administered to an animal to achieve the desired dose that will result in an increase in lean meat and an improvement in the lean meat to fat ratio. In addition, implants can also be administered intermittently during the treatment of animals to maintain an appropriate level of drug in the animal's body.

The invention has several useful features for veterinary medicine. For the pet owner or veterinarian who wishes to keep the pet lean and/or rid the pet of unwanted fat, the present invention provides a means by which this can be achieved. For poultry, beef and pork producers, the method of the invention results in leaner animal meat that should be sold at higher selling prices.

EXAMPLES

Unless otherwise noted, starting materials are generally available from commercial sources such as Aldrich Chemicals Co. (Milwaukee, WI), Lancaster Synthesis, Inc. (Windham, NH), Acros Organics (Fairlawn, NJ), Maybridge Chemical Company, Ltd. (Cornwall, England) and Tyger Scientific (Princeton, NJ). Some standard abbreviations and abbreviations are used in the description, such as AcOH (acetic acid), DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), CDI (1,1'-carbonyldiimidazole), DCM (dichloromethane) , DEA (diethylamine), DIPEA (N, N-diisopropylethylamine), DMAP (4-dimethylaminopyridine), DMF (N, N'-dimethylformamide), DMSO (dimethyl sulfoxide), EDCI (N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide), Et ₂ O (diethyl ether), EtOAc (ethyl acetate), EtOH (ethanol), G or g (gram), HATU (hexafluorophosphate methanamine 2-(1H-7-azabenzotriazol-1-yl)-1,1,3 ,3-tetramethyluronium), HBTU (O-benzotriazol-1-yl-N, N,N',N'-tetramethyluronium hexafluorophosphate), HOBT (1-hydroxybenzotriazole), Hour. or hour. (hour), IPA (isopropyl alcohol), KHMDS (potassium hexamethyldisilazane), MeOH (methanol), L or L (liter), ml (milliliter) MTBE (tert-butyl methyl ether), mg (milligram), NaBH(OAc) ₃ (sodium triacetoxyborohydride), NaHMDS (sodium hexamethyldisilazane), NMP (N-methylpyrrolidone), RH (relative humidity), RT or RT (room temperature, which is equal to ambient temperature (about 20 to 25°C)), SEM ([ 2-(trimethylsilyl)ethoxy]methyl), TEA (triethylamine), TFA (trifluoroacetic acid), THF (tetrahydrofuran) and T ₃ P (propane phosphoric anhydride).

Spectra ¹ H nuclear magnetic resonance (NMR) in all cases correspond to the proposed structures. Characteristic chemical shifts (δ) are given in parts per million (ppm) relative to the residual proton signal in the deuterated solvent (CHCl ₃ at 7.27 ppm; CD ₂ HOD at 3.31 ppm) and are described with using standard abbreviations for major peaks; for example, c is a singlet; d - doublet; t - triplet; kv - quartet; m - multiplet; br. - broadened.

ssNMR means the NMR spectrum of a substance in the solid state.

XRD stands for X-ray powder diffraction.

The term "substantially the same" when used to describe X-ray powder diffraction patterns means diffraction patterns that include peaks at diffraction angles that are within a standard deviation of +/- 0.2° 2Θ.

The term "substantially pure" when used in relation to a particular crystalline form means that said crystalline form comprises less than 10%, preferably less than 5%, preferably less than 3%, preferably less than 1%, by weight of any other physical form of Compound A or Compound D.

The reactions are usually carried out in air or, when oxygen or moisture sensitive reagents or intermediates are used, in an inert atmosphere (nitrogen or argon). If necessary, the reactors are dried under dynamic vacuum using a heat gun and anhydrous solvents (Sure-Seal™ products from Aldrich Chemical Company, Milwaukee, Wisconsin or DriSolv™ products from EMD Chemicals, Gibbstown, NJ). Commercial solvents and reagents were used without further purification. When indicated, heating of the reaction mixtures is carried out using microwave radiation from the Biotage Initiator or Personal Chemistry Emrys Optimizer. The progress of the reactions is monitored using thin layer chromatography (TLC), liquid chromatography-mass spectrometry (LCMS), high performance liquid chromatography (HPLC) and/or gas chromatography-mass spectrometry (GCMS). TLC is carried out on pre-coated silica gel plates with a fluorescence indicator (with an excitation wavelength of 254 nm) and visualization under UV light and/or with I ₂ , KMnO ₄ , CoCl ₂ , phosphomolybdic acid and/or cerium ammonium molybdate. LCMS data were obtained on an Agilent 1100 Series instrument with a Leap Technologies autosampler, Gemini C18 columns, and using MeCN/water gradients and TFA, formic acid, or ammonium hydroxide modifiers. The column eluent is analyzed using a Waters ZQ mass spectrometer scanning in both positive and negative ion modes from 100 to 1200 Da. Other similar tools are also used. HPLC data are obtained on an Agilent 1100 Series instrument using Gemini or XBridge C18 columns, MeCN/water gradients, and TFA or ammonium hydroxide modifiers. GCMS data were obtained using a Hewlett Packard 6890 drying chamber with an HP 6890 injector, an HP-1 column (12 m x 0.2 mm x 0.33 µm) and helium as carrier gas. The sample is analyzed on an HP 5973 mass selective detector scanning from 50 to 550 Da using electron ionization. Purification is performed by medium performance liquid chromatography (MPLC) using Isco CombiFlash Companion, AnaLogix IntelliFlash 280, Biotage SP1, or Biotage Isolera One instruments pre-equipped with Isco RediSep or Biotage Snap silica cartridges. Chiral purification is performed by chiral supercritical liquid chromatography (supercritical liquid chromatography - SLC) using Berger or Thar apparatuses; columns ChiralPAK-AD, -AS, -IC, Chiralcel-OD or -OJ; and mixtures of CO ₂ with MeOH, EtOH, iPrOH or MeCN or modified with TFA or iPrNH ₂ . UV detection is used to initiate the collection of fractions.

Mass spectrometry data is recorded using LCMS analysis. Mass spectrometric (MS) analysis is performed using chemical ionization at atmospheric pressure (a™ospheric pressure chemical ionization - APCI), electrospray ionization (ESI), electron impact ionization (EI) or electron scattering (electron scatter-ES). The chemical shifts of proton nuclear magnetic resonance ( ¹ H NMR) spectroscopy downfield relative to tetramethylsilane are presented in ppm and recorded on Varian spectrometers at 300, 400, 500, or 600 MHz. Chemical shifts are expressed in parts per million (ppm, δ) relative to the residual peaks of the deuterated solvent. Peak shapes are described by the following abbreviations: s - singlet; d - doublet; t - triplet; kv - quartet; quin - quintet; m - multiplet; br. c - broadened singlet; predp. - supposed. SLC analytical data is obtained using a Berger analytical instrument as described above. Optical rotation data are acquired on a PerkinElmer model 343 polarimeter using a 1 dm cuvette. Silica gel chromatography is performed primarily using Biotage or ISCO medium pressure systems and columns prepacked from various commercial vendors including Biotage and ISCO. Microanalyses are performed by Quantitative Technologies Inc. and the values obtained differ from the calculated values by no more than 0.4%.

Unless otherwise noted, chemical reactions are carried out at room temperature (about 23 degrees Celsius).

The names of the compounds and intermediates described below were compiled using the method provided by ChemBioDraw Ultra, Version 12.0 (CambridgeSoft Corp., Cambridge, Massachusetts). The naming method provided by ChemBioDraw Ultra, Version 12.0 is well known to those skilled in the art, and the naming method provided by ChemBioDraw Ultra, Version 12.0 is considered to comply with the IUPAC (International Union for Pure and Applied Chemistry) recommendations regarding organic nomenclature. chemistry, and CAS Index rules. Unless otherwise noted, all reagents are obtained from commercial suppliers and used without further purification or synthesized using methods described in the technical literature.

The terms "concentrate, "evaporate" and "concentrate in vacuo" refer to the removal of the solvent under reduced pressure on a rotary evaporator with a bath temperature of less than 60°C. Abbreviations "min." and "hour." are used to denote "minutes" and "hours", respectively. The abbreviation "TLC" means thin layer chromatography, the term "room temperature or ambient temperature" means a temperature in the range from 18 to 25°C, the abbreviation "GCMS" means gas chromatography-mass spectrometry, the abbreviation "LCMS" means liquid chromatography-mass spectrometry, the abbreviation "UHPLC" means ultra high performance liquid chromatography, the abbreviation "HPLC" refers to high pressure liquid chromatography, the abbreviation "SLC" means supercritical liquid chromatography.

The hydrogenation can be carried out in a Parr mixing reactor under hydrogen gas pressure or in a Thales-nano H-Cube flow hydrogenation apparatus filled with hydrogen at a flow rate of 1-2 ml/min. at the specified temperature.

The retention times of HPLC, UHPLC, LCMS, GCMS and SLC are measured using the methods specified in the methods.

Preparation of Intermediates and Example Compounds

Example 1 (DGAT2i-Compound/Compound D): (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine -5-carboxamide

Stage 1: 3-ethoxypyridine

Cesium carbonate (12 mol, 1.5 equiv.) and ethyl iodide (9.7 mol, 1.2 equiv.) are added to a solution of 3-hydroxypyridine (8.10 mol, 1.0 equiv.) in acetone (12 L) at 15°C. The reaction mixture is stirred at room temperature for 24 hours. The reaction mixture is filtered and the organic layer is concentrated to give the crude product. Add ethyl acetate (20 L) and wash the mixture with water (3×5 L). The organic layer was dried over sodium sulfate, filtered and concentrated to give 3-ethoxypyridine (620 g, 62%) as an oil. ¹ H NMR (400 MHz, CDCl ₃ ) δ 1.44 (t, 3H), 4.07 (q, 2H), 7.15-7.23 (m, 2H), 8.20 (dd, 1H) , 8.30 (d, 1H).

Stage 2: 3-ethoxypyridine-1-oxide

m-Chloroperoxybenzoic acid (6.5 mol, 1.3 equiv.) was added to a solution of 3-ethoxypyridine (5.0 mol, 1.0 equiv.) in dichloromethane (12 L) at 10°C. The reaction mixture is stirred at room temperature for 24 hours. Sodium thiosulfate (4 kg in 5 L of water) is added. The reaction mixture is stirred at 15°C for 2 hours. Add another portion of sodium thiosulfate (1.5 kg, in 5 L of water). The reaction mixture was stirred at 15°C for 1 hour. The mixture is extracted with dichloromethane (16 x 10 L). The combined organic layers are concentrated to give the crude product. The crude product was purified by chromatography (silica gel, dichloromethane:methanol; 100:1-10:1) to give the title compound (680 g, 97%) as a brown oil. The product was further purified by trituration with petroleum ether (4 L) at room temperature for 24 hours to give 3-ethoxypyridine-1-oxide (580 g, 83%) as a yellow solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 1.41 (t, 3H), 4.02 (q, 2H), 6.84 (dd, 1H), 7.12 (dd, 1H), 7.85 (d, 1H), 7.91-7.95 (m, 1H).

Stage 3: 2-((5-bromopyridin-3-yl)oxy)-3-ethoxypyridine

This reaction is carried out using five parallel downloads.

Diisopropylethylamine (2.69 mol, 3.7 equiv.) and bromtripyrrolidinophosphonium hexafluorophosphate (0.93 mol, 1.3 equiv.) are added with stirring to a solution of 3-ethoxypyridine-1-oxide (0.72 mol, 1.0 equiv. .) and 3-bromo-5-hydroxypyridine (0.72 mol, 1.0 equiv.) in tetrahydrofuran (2500 ml) at room temperature. The reaction mixture is stirred at room temperature for 2 days, then the individual batches are combined into a single mass. The resulting suspension is concentrated to dryness and the residue is taken up in dichloromethane (25 L). The organic layer was washed with 1N sodium hydroxide (15 L), water (3 x 20 L) and brine (20 L). The organic layer is dried over sodium sulfate, filtered and concentrated to give an oil. The crude oil was purified by column chromatography (silica gel, petroleum ether:ethyl acetate; 10:1-1:1) to give the crude product as a brown solid. The resulting solid was triturated with methyl t-butyl ether:petroleum ether (1:10; 11 L) to give 2-((5-bromopyridin-3-yl)oxy)-3-ethoxypyridine (730 g, 69%) as a yellowish solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 1.49 (t, 3H), 4.16 (q, 2H), 7.04 (dd, 1H), 7.25 (dd, 1H), 7.68 -7.73 (m, 2H), 8.44 (d, 1H), 8.49 (d, 1H). MS (ES+) 297.1 (M+H).

Stage 4: ethyl 2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)pyrimidine-5-carboxylate

A solution of 2-((5-bromopyridin-3-yl)oxy)-3-ethoxypyridine (300 mmol, 1.0 equiv.) in tetrahydrofuran (1.3 L) was degassed with nitrogen for 30 minutes. Turbo Grignard (390 mmol, 1.3 equiv., 1.3 M in tetrahydrofuran) is added at room temperature at a rate at which the temperature of the mixture remains below 30°C. The reaction mixture is allowed to cool to room temperature and the mixture is stirred for 3 hours. The reaction mixture is cooled to 10°C and added to the mixture of zinc chloride (390 mmol, 1.3 equiv., 1.9 M in 2-methyltetrahydrofuran) at a rate at which the temperature of the mixture remains below 15°C. The resulting suspension is heated to room temperature to completely dissolve the precipitate and then cooled again to 10°C. Ethyl 2-chloropyrimidine-5-carboxylate (360 mmol, 1.2 equiv) and dichloro[bis(2-(diphenylphosphino)phenyl)ether]palladium (II) (6.00 mmol, 0.02 equiv) are added to the mixture. .) in the form of solids. The resulting suspension is degassed with nitrogen for 30 minutes, then heated to 50°C and maintained at this temperature for 16 hours. The reaction mixture is treated under aqueous conditions, then heated with successive additions of sodium ethylenediaminetetraacetic acid, silicon sulfide (thiosilica) and carbon to remove metal impurities. The crude product is recrystallized from methanol (450 ml) to give ethyl 2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)pyrimidine-5-carboxylate (77 g, 70%) as pale yellow solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 1.44 (t, 3H), 1.50 (t, 3H), 4.19 (q, 2H), 4.46 (q, 2H), 7.00 -7.04 (m, 1H), 7.25 (s, 1H), 7.71 (d, 1H), 8.59 (s, 1H), 8.66 (d, 1H), 9.32 ( s, 2H), 9.55 (s, 1H),

Step 5: 2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)pyrimidine-5-carboxylic acid (Intermediate 1)

Sodium hydroxide (307 mmol, 1.5 equiv., 4M aqueous solution) and methanol (50 mL) are added to a suspension of 2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)pyrimidin- 5-carboxylate (205 mmol, 1.0 equiv.) in tetrahydrofuran (300 mL). The resulting solution was stirred at room temperature for 3 hours. The reaction mixture was diluted with water (400 ml) and extracted with 2:1 diethyl ether:heptanes (2×300 ml). The aqueous layer was acidified to pH 4 with 4M hydrochloric acid. The resulting suspension is stirred at room temperature for 1 hour. The solid was collected by filtration, washed with water and dried to give 2-(5-((3-ethoxy-pyridin-2-yl)oxy)-pyridin-3-yl)-pyrimidine-5-carboxylic acid (69 g, 100%) as a solid pale yellow substances. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.37 (t, 3H), 4.18 (q, 2H), 7.19 (dd, 1H), 7.58 (dd, 1H), 7 .70 (dd, 1H), 8.35-8.40 (m, 1H), 8.66 (d, 1H), 9.33 (s, 2H), 9.41 (d, 1H), 13, 9 (br. s, 1H).

Step 6: (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide (Example 1 (DGAT2i -compound))

Oxalyl chloride (13.8 ml, 160 mmol, 1.2 equiv.) and dimethylformamide (0.510 ml, 6.65 mmol, 0.05 equiv.) are added to a suspension of 2-(5-((3-ethoxypyridin-2-yl )oxy)pyridin-3-yl)pyrimidine-5-carboxylic acid (45.0 g, 133 mmol, 1.0 equiv.) in dichloromethane (500 mL). The suspension is stirred the mixture for 2 hours to obtain a solution. The reaction mixture was concentrated to give the crude acid chloride as a red solid. A solution of (S)-tetrahydrofuran-3-amine (12.2 g, 140 mmol, 1.05 equiv.) and diisopropylethylamine (51.0 ml, 293 mmol, 2.2 equiv.) in tetrahydrofuran (100 ml) dropwise added to a solution of crude acid chloride in dichloromethane (200 ml) at 0°C. The reaction mixture is allowed to warm to room temperature and the mixture is stirred for 16 hours. Water (1.0 L) and ethyl acetate (600 ml) were added to the mixture, the organic layer was separated, washed with saturated sodium hydrogen carbonate solution, dried over magnesium sulfate and filtered. The filtrate is treated with activated charcoal (20 g) and stirred at 65° C. for 20 minutes. The warm suspension is filtered and the filtrate is concentrated to give a pale yellow solid which is recrystallized from a mixture of methanol and ethyl acetate (1:4, 1 L) to give (S)-2-(5-((3-ethoxypyridin-2-yl )oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide (43.5 g, 81%) as a colorless solid. The title compound was combined with products (108.7 g, 266.8 mmol) obtained in the same way, suspended in ethyl acetate (1.0 L) at 80° C. and kept under the same conditions for 4 hours. The suspensions are allowed to cool to room temperature and the mixture is stirred for 4 days. The solid product is collected by filtration, washed with ethyl acetate (3×200 ml) and dried in high vacuum at 50°C for 24 hours to obtain (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridine -3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide (100.5 g, 92%) as a colorless solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 1.38 (t, 3H), 1.89-1.98 (m, 1H), 2.15-2.26 (m, 1H), 3, 65(dd, 1H), 3.70-3.78(m, 1H), 3.85-3.92(m, 2H), 4.18(q, 2H), 4.46-4.55( m, 1H), 7.18 (dd, 1H), 7.58 (dd, 1H), 7.69 (dd, 1H), 8.37 (dd, 1H), 8.64 (d, 1H), 8.95 (d, 1H), 9.28 (s, 2H), 9.39 (d, 1H). MS (ES+) 408.4 (M+H). Melting point 177.5°C. Elemental analysis for C ₂₁ H ₂₁ N ₅ O ₄ : calculated C 61.91; H5.20; N 17.19; found C 61.86; H 5.18; N 17.30.

The solid obtained by this procedure is characterized by powder X-ray diffraction (XRD) and is defined as form 1 of compound D.

Alternative Step 6 for Preparation of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide (Example Compound D one)

A 100 ml reactor was charged with acetonitrile (35 ml), 2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)pyrimidine-5-carboxylic acid (5.0 g, 15 mmol) and (S)-tetrahydrofuran-3-amine hydrochloride (2.2 g, 18 mmol, 1.2 equiv.). Add diisopropylethylamine (18 ml, 103 mmol, 7.0 equiv.), maintaining the temperature in the range from 20°C to 30°C. A solution of propane phosphoric anhydride (T3P) in acetonitrile (21 ml, 30 mmol, 2.0 equiv.) is loaded at a rate at which the temperature of the mixture remains below 45°C. The reactor is heated to 40±5°C and maintained at the same temperature for 1 hour, then a sample of the reaction mixture is taken to monitor the progress of the reaction. The reaction mixture is cooled to a temperature in the range from 20°C to 25°C and add tetrahydrofuran (25 ml). Load sodium bicarbonate solution (0.5 M, 40 ml) and the mixture is stirred for 1 hour. Check the pH value, which is 8.5. Add ethyl acetate (40 ml) and stir the mixture for 15 minutes. The mixture is left and the phases are separated. The aqueous layer was transferred to a separating funnel and extracted again with ethyl acetate (100 ml). The organic phases are combined and the mixture is washed with water (40 ml). The organic layer is transferred to a 100 ml reactor in batches and concentrated in vacuo to low volume. Methyl ethyl ketone (100 ml) was added and the mixture was concentrated to a final volume of approximately 60 ml. The vacuum is removed, the slurry is refluxed until the solid particles are washed off the walls of the reactor. The suspension is cooled to 15° C. over 2 hours and granulated overnight. The solid is isolated by filtration, the reactor and the precipitate are washed twice with methyl ethyl ketone (10 ml each). The solids are dried in a vacuum oven at 50° C. to give 4.86 g (81%) of the expected product. The solid product obtained by this procedure is characterized by powder X-ray diffraction and is designated Form 2 of Compound D.

Conversion of Compound D Form 2 to Compound D Form 1

Form 2 (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide is loaded into a 100 ml reactor (example 1) (10.0 g, 24.6 mmol, 1.00 equiv.), methyl ethyl ketone (8.8 ml/g, 88.0 ml) and water (1.2 ml/g, 12.0 ml ). The reactor is heated to 50°C for 30 minutes. Complete dissolution is observed at a temperature of about 44°C. The reactor is cooled to 40° C. for 30 minutes, then Form 1 (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3) is seeded -yl)pyrimidine-5-carboxamide (Example 1 compound D) (0.050 g, 0.123 mmol, 0.0050 equiv.). After inoculation, the cloudy suspension is stirred for 1 hour, after which it is cooled to 5°C for 2 hours and then stirred at 5°C for 12 hours. During mixing, a control sample was taken and analyzed using XRD to confirm that the solids were form 1 of compound D. ml). The solid product is dried in a vacuum oven at 50°C to obtain 8.15 g (81.5%) of the desired product. The PRDH of the target product corresponds to form 1 of compound D.

Powder X-ray diffraction pattern (XRD)

X-ray powder diffraction analysis was carried out using a Bruker AXS D8 Advance diffractometer with a copper source (Kα average wavelength 1.54056Å) equipped with a primary double Gobel mirror. Diffracted radiation is recorded using a PSD-Lynx Eye detector. The primary and secondary devices are equipped with 2.5 Soller slots. The voltage and current of the X-ray tube are set to 40 kV and 40 mA, respectively. Data is collected in a Theta-Theta goniometer in a closed dual scan from 3.0 to 40.0 degrees 2-theta with 1000 steps and a scan rate of 6 seconds per step. Samples are placed in a low background silicon sample holder (C79298A3244B261). Data is collected using Bruker DIFFRAC Plus software. Data analysis is performed using the EVA DIFFRACT Plus software.

The TXR data file is not processed prior to searching for peaks. Using the EVA software peak search algorithm, peaks with a threshold value of 5 and a width of 0.2 are selected. The automated value output is visually checked for plausibility and adjusted manually if necessary. Usually choose peaks with a relative intensity ≥3%. Peaks that have not been isolated and that correspond to noise interference are also not taken into account. The typical error associated with the position of the peak in the USP claimed XRD is within +/- 0.2° (USP-941).

Table 1. Principal peaks on PRDH characterizing the crystalline compounds of example 1 (compounds D)

Form 1 of the compound of example 1 Form 2 of the compound of example 1 Angle 2Θ (°) Angle 2Θ (°) 5.3, 7.7, 15.4 6.5, 9.3, 13.6

Figure 1 shows the characteristic X-ray powder diffraction pattern of the crystalline form 1 of the compound of Example 1 (compound D) (vertical axis: intensity (pulses per second); horizontal axis: two theta angle (degrees).

Figure 2 shows the characteristic X-ray powder diffraction pattern of the crystalline Form 2 of the compound of Example 1 (compound D) (vertical axis: intensity (pulses per second); horizontal axis: two theta angle (degrees)).

Example 2 Preparation of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridine-2- yl)benzoic acid (compound A (AKKi-compound))

It should be noted that in the preparation of Compound A in some of the processes described herein, it may be necessary to protect the removed functionality (eg, primary amine, secondary amine, carboxyl group in Formula I precursors). The need for such protection will vary depending on the nature of the removed functionality and the conditions of the acquisition methods. The need for such protection is easily determined by a person skilled in the art. The use of such methods of introduction/removal of protection is also within the purview of a person skilled in the art. For a general description of protecting groups and their uses, see T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991. In addition, the present invention is not limited to the specific synthesis methods presented in the description, which may vary.

Intermediate A1: 1-isopropyl-4,6-dihydrospiro[indazol-5,4'-piperidine]-7(1H)-one hydrochloride salt

Stage 1: tert-butyl-9-oxo-3-azaspiro[5.5]undec-7-ene-3-carboxylate

In a dry reactor load tert-butyl-4-formylpiperidine-1-carboxylate (108 kg), cyclohexane (1080 l) and pyrrolidine (64.8 kg) at 25-30°C. The mixture is stirred for 5-10 minutes and then refluxed for 12-16 hours, collecting water using a Dean-Stark trap. The reaction mixture is cooled to 50-60° C. and vacuum is applied at this temperature to distill off excess pyrrolidine and cyclohexane. The reaction mixture is cooled to 25-30° C. and cyclohexane (648 L) and methyl vinyl ketone (49.63 kg) are added successively. The mixture is stirred for 12-16 hours, then filtered and the filtrate is loaded into a clean dry reactor. The solution is cooled to 10-15°C, then a solution of acetic acid (54.75 kg) in water (54 l) is slowly added, keeping the temperature below 15°C. After the addition, the mixture is heated to 25-30° C. and stirred for 12-16 hours. The layers are separated and the aqueous layer is extracted with ethyl acetate (324 L). The combined organic layers are washed with a solution of sodium bicarbonate (32.34 kg) in water (324 L), then dried over sodium sulfate. The solids are washed with ethyl acetate (54 l) and the combined filtrates are concentrated under reduced pressure at a temperature below 40°C. The reactor is charged with n-heptane (216 L) and the solvent is distilled off to dryness under reduced pressure at a temperature below 40°C. The mixture is cooled to 25-30° C. and n-heptane (216 L) is charged to the reactor. The mixture is stirred for 1-2 hours after the formation of a solid precipitate. The solid product is then collected by filtration, washed with n-heptane (54 L) and dried at 40-50°C for 10-12 hours to obtain the desired product (90.1 kg, 67% yield).

Stage 2: (E)-tert-butyl-10-((dimethylamino)methylene)-9-oxo-3-azaspiro[5.5]undec-7-ene-3-carboxylate

A clean dry reactor is charged with tert-butyl 9-oxo-3-azaspiro[5.5]undec-7-en-3-carboxylate (50 kg), N, N-dimethylformamide (500 L) and N, N-dimethylformamide-dimethyl acetal (135 kg) at 25-30°C in a nitrogen atmosphere. The reaction mixture is stirred for 5-10 minutes, then heated to 120-130°C and maintained at the same temperature for 20 hours. The mixture is then cooled to 50-60°C and the solvent is distilled off in high vacuum at a temperature below 60°C. A mixture of xylenes (200 l) is loaded at a temperature below 45°C and the solvent is distilled off in high vacuum at a temperature below 60°C. This operation is repeated with another load of xylenes (200 l). Then toluene (200 l) is charged into the reactor and the solvent is distilled off in high vacuum at a temperature below 60°C. The operation is repeated with a second charge of toluene (200 l). Methyl tert-butyl ether (100 L) is then charged at a temperature below 30°C and the solvent is distilled off under high vacuum at a temperature below 40°C. The mixture is cooled to 15-20°C and download methyl tert-butyl ether (100 l) at a temperature below 20°C. The mixture is stirred for 20-30 minutes, the solid is collected by filtration, washed with methyl tert-butyl ether (50 L) and dried without applying vacuum at 50-55°C for 10 hours to obtain the target compound (52.1 kg, yield 87%). ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 7.48 (s, 1H), 6.57 (d, J=9.97 Hz, 1H), 5.99 (d, J=10.16 Hz, 1H), 3.32-3.51 (m , 4H), 3.06 (s, 6H), 2.72 (s, 2H), 1.57-1.66 (m, 2H), 1.41-1.53 (m, 11H).

Stage 3: tert-butyl-1-isopropyl-1,4-dihydrospiro[indazole-5,4'-piperidine]-1'-carboxylate

(E)-tert-Butyl-10-((dimethylamino)methylene)-9-oxo-3-azaspiro[5.5]undec-7-ene-3-carboxylate (80 kg), toluene (704 L ) and trimethylamine (16 l) at 25-30°C. The reaction mixture is heated to 70-80°C and a solution of the hydrochloride salt of isopropylhydrazine in methanol (1.25 equiv., 141 kg) is added within 4-5 hours. The reaction mixture is then stirred for 8-10 hours at 70-80°C and cooled to 15-25°C. Then slowly add a solution of citric acid (48 kg) in water (480 l), maintaining the internal temperature below 25°C. Ethyl acetate (208 L) is added and the mixture is stirred for 10 minutes. The layers are separated, the organic layer is washed successively with a solution of citric acid (48 kg) in water (480 L) and then only with water (320 L). The combined aqueous layers are extracted with ethyl acetate (320 L). The combined organic layers are dried over sodium sulfate (8 kg) and the solvents are evaporated to dryness under reduced pressure at a temperature below 40°C. Dichloromethane (240 L) was charged to the reactor and the mixture was stirred at 25-30° C. until a clear solution was obtained. Activated carbon (1.84 kg), magnesium silicate (1.84 kg) and silica gel (32 kg, 100-200 meesh) are sequentially charged at 25-30° C. and the heterogeneous mixture is stirred for 1 hour. The slurry is then filtered on a Hyflow bed made by mixing Hyflow supercell (8 kg) and dichloromethane (40 L). The filter cake is washed with dichloromethane (three times 120 l). The combined filtrates are reloaded into the reactor and the solvent is evaporated off under reduced pressure below 40°C. n-heptane (160 l) is loaded into the reactor and distilled off under reduced pressure at a temperature below 40°C. n-heptane (200 l) is loaded into the reactor and the mixture is cooled to 0-5°C. After stirring for 12-15 hours, the solid is collected by filtration at 0°C, washed with chilled (0-5°C) n-hethane (160 L) and dried in vacuo at 40-50°C to give the title compound ( 82.4 kg, 75%). ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 7.25 (s, 1H), 6.42 (dd, J=10.05, 0.49 Hz, 1H) 5.84 (d, J=9.95 Hz, 1H), 4.42-4, 52 (m, 1H), 3.36-3.53 (m, 4H), 2.62 (s, 2H) 1.56-1.68 (m, 2H) 1.45-1.55 (m, 17H).

Step 4: 1-isopropyl-4,6-dihydrospiro[indazol-5,4'-piperidine]-7(1H)-one hydrochloride salt

In a clean dry reactor at 25-30°C load tert-butyl-1-isopropyl-1,4-dihydrospiro[indazole-5,4'-piperidine]-1'-carboxylate (60 kg) and methanol (600 l). N-bromosuccinimide (32.4 kg) was added to the mixture in five portions over 30-40 minutes at 25-30° C. and stirring was continued for 30-60 minutes. A solution of sodium thiosulfate pentahydrate (5.4 kg) in water (102 L) was slowly added to the mixture, keeping the internal temperature of the mixture below 30°C. The mixture is stirred for 20-30 minutes, then the solvent is evaporated under reduced pressure at a temperature below 45°C. The residue is cooled to 25-30° C. and the reactor is charged with 2-methyltetrahydrofuran (420 L) and water (90 L). The mixture is stirred for 15-20 minutes, then the layers are separated and the aqueous layer is further extracted with 2-methyltetrahydrofuran (120 L). The combined organic extracts are treated for 15-20 minutes at 25-30°C with a solution of sodium hydroxide (4.8 kg) in water (120 l). The layers are separated, the organic layer is washed with water (120 L), a solution of sodium chloride (12 kg) in water (120 L) and then dried over sodium sulfate (6 kg). The mixture is filtered, the filter cake is washed with 2-methyltetrahydrofuran (30 L) and the combined filtrate is reloaded into the reactor. The solvent is completely distilled off at a temperature below 45°C under reduced pressure and the residue is redissolved in tetrahydrofuran (201 l). In another clean dry reactor at 25-30° C., potassium tert-butoxide (60.6 kg) and tetrahydrofuran (360 L) are charged. A solution of the residue in tetrahydrofuran was slowly added to the resulting mixture, keeping the temperature below 30°C. The reaction mixture is heated to 60-65°C and kept at the same temperature for 1-2 hours. After completion of the reaction, the mixture is cooled to 0-10°C and slowly quenched with a solution of hydrochloric acid (1 N, 196 l), maintaining the internal temperature below 10°C. The reaction mixture is allowed to warm to 25-30° C. and ethyl acetate (798 L) is added. After stirring for 15-20 minutes, the layers are separated and the aqueous layer is further extracted with ethyl acetate (160 L). The combined organic layers are washed with water (160 L), dried over sodium sulfate (8 kg), filtered and the filter cake washed with ethyl acetate (300 L). The solvents are completely distilled off under reduced pressure at temperatures below 45° C., ethyl acetate (540 l) at 25-30° C. and then methanol (156 l) are charged into the reactor. The mixture is cooled to 0-5°C and acetyl chloride (79.8 kg) is slowly added at the same temperature, maintaining the temperature in the indicated range. After that, the mixture is allowed to warm up to 20-25°C and the mixture is stirred at the same temperature for 4-5 hours. The resulting suspension is filtered, the solid product is washed with ethyl acetate (120 l), then dried at 40-45°C for 8-10 hours, obtaining a crude product (33.5 kg, 65%).

The final purification step is carried out by solubilization of the obtained solid product (56.8 kg) in methanol (454.4 l) in a clean dry reactor at 25-30°C. The solution is stirred for 30-45 minutes, then loaded through a cartridge filter (0.2 microns) into a clean dry reactor at 25-30°C. The methanol is distilled off under reduced pressure at a temperature below 50°C until ~1 vol. solvent. The reaction mixture is cooled to 25-30° C. and fresh acetonitrile (113.6 L) is charged through a cartridge filter (0.2 micron). The solvents are distilled off under reduced pressure at a temperature below 50°C until ~1 vol. solvent. The reaction mixture is cooled to 25-30°C and loaded into the cutter through a cartridge filter (0.2 micron) fresh acetonitrile (190 l). The mixture is heated to 65-70°C and stirred for 45 minutes, then cooled to 25-30°C and stirred for 1 hour. The resulting suspension is filtered, the filter cake is washed with cold (15° C.) acetonitrile (56.8 L). The solid product is dried under reduced pressure at 40-50°C for 8 hours to obtain intermediate compound A1 (36.4 kg, 64%). ¹ H NMR (400 MHz, CD ₃ OD) δ ppm 7.43 (s, 1H), 5.32-5.42 (m, 1H), 3.15-3.25 (m, 4H), 2.89 (s, 2H), 2.64 (s, 2H), 1.69-1.90 (m, 4H), 1.37-1.45 (m, 6H); ESI [M+H] ⁺ = 248.

Intermediate A2: 2-(4-(tert-butoxycarbonyl)phenyl)-6-methoxyisonicotinic acid

A clean, dry reactor is charged with 2,6-dichloroisonicotinic acid (30 kg) and methanol (120 L) at 20-25°C. The suspension is stirred for 5 minutes and then heated to 65° C. (reflux temperature). A solution of sodium methoxide in methanol (30%, 87.2 kg) was slowly added to the mixture via an addition funnel over at least 4 hours. The funnel is washed with methanol (15 L) and the mixture is stirred at 65° C. for at least 15 hours. The mixture is cooled to 45°C and the solvents are distilled off under reduced pressure to a final volume of ~90 l. A solution of potassium bicarbonate (28.2 kg) and potassium carbonate (21.6 kg) in water (180 L) at 40-45°C is added to the reactor. The reactor containing the aqueous solution was washed with water (21 L) and the washing liquid was charged to the reaction mixture. The mixture is distilled off under reduced pressure at a temperature below 80°C to a final volume of ~240 l, then cooled to 20-25°C.

Another clean dry reactor is charged with tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxoborolan-2-yl)benzoate (52.3 kg) and dioxane (340 kg) and mixed mixture at 2-25°C until complete dissolution. The contents of the first reactor are then heated to 40° C. until completely dissolved and the solution is transferred to this new reactor. The reaction mixture is cooled to 20-25°C and perform the operation of deoxygenation, cyclically creating a vacuum and filling with nitrogen. The mixture is cooled to 0-10°C, the reactor is loaded with palladium acetate (0.65 kg) and then triphenylphosphine (2.46 kg) in a stream of nitrogen. The mixture is heated to 20-25°C and the deoxygenation operation is performed again, cyclically repeating the creation of a vacuum and filling the reactor with nitrogen. The mixture is heated to 80°C and maintained at the same temperature for at least 18 hours. The mixture is cooled to 20-25° C., then methyl tert-butyl ether (133.2 kg) and water (30 L) are added sequentially to the reactor. The layers are separated, the aqueous layer is diluted with water (110 L) and then extracted with methyl tert-butyl ether (110 L). The combined organic extracts are washed with a solution of citric acid (52 kg) in water (84 L) and the layers are separated. The aqueous layer was further extracted with methyl tert-butyl ether (88.8 kg), the organic layers were combined, then washed three times each with a third of a solution of sodium chloride (43 kg) in water (80 L). After a final separation of the layers, the organic layer is filtered through a pall filter containing a carbon cartridge and the filter cake is washed with methyl tert-butyl ether (11.2 kg). The filtrate is distilled off under reduced pressure below 50° C. to a volume of ~90 L and then co-distilled with heptane (120 L) at a temperature below 50° C. and a volume of ~120 L. The mixture is cooled to 20-25°C for 1 hour, then stirred at the same temperature for an additional 1 hour. The suspension is filtered, the filter cake is washed with heptane (3×18 L) and then with acetonitrile (3×18 L). The resulting wet solid is dried under vacuum and under a stream of nitrogen below 45° C. for at least 15 hours to give Intermediate A2 (44.6 kg, 87% yield). ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 8.13 (s, 2H), 8.09 (s, 2H), 7.97 (d, J=1.17 Hz, 1H), 7.34 (d, J=0.98 Hz, 1H), 4.08 (s, 3H), 1.61 (s, 9H); ESI [M+H] ⁺ = 330.

Intermediate A3: tert-butyl-4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6- methoxypyridin-2-yl)benzoate

A round bottom flask was charged with 2-(4-(tert-butoxycarbonyl)phenyl)-6-methoxyisonicotinic acid (Intermediate A2, 15.2 g, 46.2 mmol) and ethyl acetate (140 mL). To the mixture was added 1,1'-carbonyldiimidazole (8.98 g, 55.4 mmol) in one portion, and the mixture was stirred for 1 hour at room temperature. To the mixture was added 1-isopropyl-4,6-dihydrospiro[indazol-5,4'-piperidin]-7(1H)-one hydrochloride (Intermediate A1, 14.8 g, 52.2 mmol) followed by N, N -diisopropylethylamine (9.1 ml, 52.2 ml) and the reaction mixture was stirred for 18 hours at room temperature. Add aqueous 2 M HCl (40 ml) followed by the addition of 1 M potassium hydrogen sulfate (40 ml) and 50 ml of heptane. The resulting mixture was stirred for 1 hour at room temperature. The mixture is transferred to a separating funnel. The organic phase is separated, washed successively with water (20 ml), saturated sodium bicarbonate solution (30 ml), water (20 ml) and brine (20 ml), dried over 20 g of magnesium sulfate and 10 g of silica gel, filtered and concentrated in vacuum. During concentration, a solid product begins to form. The residue is taken up in 40 ml of ethyl acetate at 80° C. and heptane (120 ml) is slowly added dropwise. The mixture was stirred at 80° C. for 1 hour, then slowly cooled to room temperature with stirring for 1 hour, and the mixture was stirred for 18 hours at room temperature. The solid is collected by filtration, washed with water and ethyl acetate-heptane (1:3) and dried in vacuo at 50° C. for 18 hours to give intermediate A3 (19.64 g, 76% yield).

Alternative Preparation of Intermediate A3

A clean, dry reactor is charged with acetonitrile (219 kg) and 2-(4-(tert-butoxycarbonyl)phenyl)-6-methoxyisonicotinic acid (Intermediate A2, 34.8 kg) at 20-25°C. The mixture is stirred for 5 minutes and 1,1-carbodiimidazole (18.9 kg) is added in three successive portions. The suspension is further stirred at 20-25° C. for at least 1 hour, then 1-isopropyl-4,6-dihydrospiro[indazol-5,4'-piperidin]-7(1H)-one hydrochloride salt (intermediate A1, 33.0 kg) followed by addition of N,N-diisopropylethylamine (20.5 kg) via pump. The reagent pump as well as the reactor walls are rinsed with acetonitrile (13.7 kg) and the mixture stirred at 20-25° C. for at least 2 hours. The mixture is then seeded with crystallization of tert-butyl-4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl) -6-methoxypyridin-2-yl)benzoate (Intermediate A3, 209 g) and stir the mixture for at least 30 minutes. After confirming the onset of crystallization, a solution of citric acid monohydrate (58.5 kg) in water (257 L) is added within 1 hour. The resulting suspension is further stirred at 20-25°C for at least 2 hours, then filtered and the filter cake washed with a mixture of acetonitrile (68.4 kg) and water (87 l). Wash liquid is also used for rinsing the reactor. The solid product is dried under reduced pressure below 55° C. to give intermediate A3 (43.44 kg, 73% yield).

Compound A (free acid form): 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6 -methoxypyridin-2-yl)benzoic acid

tert-Butyl-4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6- methoxypyridin-2-yl)benzoate (3.7 g, 6.6 mmol) and toluene (25 ml). To the mixture was added dropwise with stirring 85% phosphoric acid (3.0 ml), the reaction mixture was heated to 60° C. and kept at the same temperature for 4 hours. A colorless thick resin is formed. The reaction mixture is cooled to room temperature and water is added. A white precipitate forms. The organic toluene layer is discarded, leaving an aqueous layer and a solid product. Add ethyl acetate (60 ml) and 4N NaOH solution to achieve a pH value of ~7. The layers are separated and the aqueous layer is extracted with ethyl acetate (50 ml). The combined ethyl acetate organic layers are dried over sodium sulfate, filtered and concentrated in vacuo to give the product as a white solid. The product is dissolved in ethyl acetate (80 ml) at 50° C. and heptane (90 ml) is added slowly. The heat is removed, the mixture is cooled to room temperature and stirred for 16 hours. The solid precipitate formed was collected by filtration, washed with mother liquor and dried to give the title compound (compound A as free acid, 2.15 g, 65% yield) as a white solid.

Alternative Preparation of Compound A (Free Acid Form)

A clean dry reactor is charged with acetonitrile (130.4 kg) and tert-butyl-4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine] -1'-carbonyl)-6-methoxypyridin-2-yl)benzoate (Intermediate A3, 20.72 kg) at 20-25°C. The mixture is stirred for 5 minutes, then p-toluenesulfonic acid (8.5 kg) is charged with a gentle purge of nitrogen. The reaction mixture is heated to 70°C and maintained at the same temperature for at least 6.5 hours. The mixture was then cooled to 40° C., compound A was seeded (104 g) and water (83 L) was added slowly over at least 1 hour. The mixture is further stirred at 40°C for at least 4 hours, then cooled to 20-25°C for 2 hours. The mixture is stirred for at least 2 hours, then filtered and the filter cake washed with a solution of acetonitrile (33 kg) in water (41 L). This washing liquid is also used for rinsing the reactor. The resulting solid product was dried under reduced pressure below 55° C. to give Compound A (16.5 kg, 89% yield).

Preparation of form 1 of compound A: anhydrous mono-tris salt of compound A

The flask was charged with 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl ) benzoic acid (151 mg, 0.300 mmol) and 3 ml of ethanol. The mixture is heated to 80°C for 5 minutes to dissolve the solid and then cooled to room temperature. Tris(hydroxymethyl)aminomethane (39 mg, 0.32 mmol) is added and the mixture is stirred overnight at room temperature. Heptane (2.25 ml) was added dropwise to the mixture to give a suspension, which was heated to 50° C. to give a clear solution. The mixture is cooled to room temperature overnight with stirring. A white solid precipitate formed and the mixture was stirred for an additional 3 days. The precipitate was collected by filtration and dried in a vacuum oven at 50° C. overnight to give Form 1 (151 mg, 0.242 mmol, 81% yield).

Alternative Preparation of Compound A Form 1: Anhydrous Mono-Tris Salt of Compound A

Ethanol (83 L) was charged to a clean, dry reactor, followed by the addition of Compound A (9.43 kg) and Tris (2.55 kg), maintaining the temperature of the mixture at 20-25°C. The walls of the reactor are rinsed with ethanol (2 l), the resulting mixture is heated to 65-70°C, maintaining the specified temperature for at least 30 minutes until the solids are completely dissolved, then cooled to 45-50°C. The warm mixture is filtered through a 10 μm in-line propylene filter and the reactor and filter are washed with ethanol (9 L). n-heptane (24 l) is loaded into a warm solution through the same in-line filter and the mixture is seeded with the crystallization of anhydrous tris-salt 4-(4-(1-isopropyl-7-oxo-1,4,6,7- tetrahydrospiro[indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid (100 g) in ethanol (0.5 l) at 45-50°C. The specified temperature is maintained for at least 2 hours before cooling to 20-25°C for at least 2 hours. The mixture is stirred for at least 5 days. The suspension is then filtered and the filter cake washed with a mixture of ethanol (13 L) and n-heptane (6 L). The solid product is dried under reduced pressure at a temperature below 45°C for at least 12 hours to obtain the compound of example 1 (11.7 kg, 77%).

Preparation of form 2 of compound A: mono-tris salt trihydrate of compound A

Compound A Form 2 was prepared from Compound A Form 1. Compound A Form 1 (1.7214 g, 2.760 mmol), isopropanol (16.50 ml, 215.8 mmol) and water (688 µl, 38.190 mmol). The mixture is stirred (300 rpm) for about 72 hours at a reactor jacket temperature of 25°C. The reaction mixture is heated to 40°C for 15 minutes, maintained at this temperature for about 24 hours, cooling once to 20°C to take a sample for testing. PRDH shows the presence of a mixture of forms; therefore, additional water is added (688 µl, 38.190 mmol). The stirring speed is increased to 400 rpm, the suspension is stirred for 6 hours and then cooled to 15°C. The solid product is collected by filtration on a 60 ml/40 M filter and washed with isopropanol/water (96/4). The resulting product is form 2 of compound A, which confirms the PRDH.

Alternative Preparation of Compound A Form 2: Compound A Mono-Tris Salt Trihydrate

Isopropanol (60.4 kg) is charged to a clean dry reactor and Compound A (16.68 kg) and Tris (4.42 kg) are added while maintaining the temperature at 20-25°C. The mixture is stirred for 5 minutes, then water (6.7 kg) is added and the suspension is heated to 55°C. The new clean solution is loaded through an in-line 10 μm polypropylene filter into a preheated clean dry reactor (50-55°C). The solution is then seeded with the crystallization of the mono-tris salt of compound A in the form of a trihydrate (167 g). After verifying that the crystallization seed is maintained, the mixture is cooled to 15°C for at least 2 hours, then maintained at 15°C for at least 16 hours. The suspension is filtered and the filter cake washed with chilled isopropanol (13.1 kg). The solid product is then dried under reduced pressure below 25° C. to give Compound A Form 2 (22.1 kg, 98% yield) as the sole product.

Compound A Form 1 is anhydrous and thermodynamically stable below about 0.2 (20% RH) water activity at ambient temperature. Form 1 XRD of Compound A is essentially the same as the XRD shown in Figure 3. The characteristic peaks of Compound A Form 1 on XRD are observed at the following 2-theta angles (2Θ±0.2°): 9.6, 10.7 and 11.3. The location of the peaks and their intensity in the PRDH presented in figure 3 are shown in table 2.

Table 2. Location of peaks in PRDH form 1 of compound A and their relative intensities

Degrees 2Θ
± 0.2° Relative intensity (%) Degrees 2Θ
± 0.2° Relative intensity (%) Degrees 2Θ
± 0.2° Relative intensity(%) 5.2 62 17.8 86 25.4 25 9.6 13 18.9 100 26.4 9 10.7 fourteen 19.5 77 26.6 eleven 11.3 eleven 20.1 eleven 27.2 9 12.1 6 21.1 29 28.3 9 13.3 60 22.2 fifteen 29.3 ten 13.9 9 22.4 19 29.9 9 14.0 ten 22.6 21 30.4 13 15.5 eleven 23.6 53 31.7 13 16.2 58 24.1 54 33.4 fifteen 17.0 27 24.5 16 5.2 62 17.8 86 25.4 25

The Raman spectrum of form 1 of compound A is essentially the same as the Raman spectrum presented in figure 4. The Raman spectrum of form 1 of compound A shows characteristic peak shifts, expressed in cm ^-1 , at 568, 698, 989, 1218, 1511, 1561 and 1615±2 cm ^-1 . The positions of the peaks (± 2 cm ^-1 ) of form 1 of compound A and their normalized intensity (W=weak, M=medium, S=strong) of the spectrum presented in figure 4 are shown in table 3.

Table 3. Location of peaks in the Raman spectrum of form 1 of compound A and their normalized intensity

Peak position
(cm ^-1 ) Normalized Intensity Peak position
(cm ^-1 ) Normalized Intensity Peak position
(cm ^-1 ) Normalized Intensity 115 M 794 W 1363 W 156 W 842 W 1388 W 170 W 885 W 1435 W 241 W 929 W 1466 W 274 W 989 W 1478 W 311 W 1011 W 1511 W 334 W 1047 W 1561 W 350 W 1071 W 1615 S 417 W 1090 W 1671 M 456 W 1119 W 2840 W 476 W 1143 W 2885 W 568 W 1169 W 2914 W 608 W 1187 W 2945 W 628 W 1196 W 2998 W 653 W 1218 W 3027 W 678 W 1244 W 3066 W 698 W 1265 W 3129 W 755 W 1315 W 779 W 1345 M

The ¹³ C ssNMR spectrum of Compound A Form 1 is essentially the same as the ¹³ C ssNMR spectrum shown in Figure 5. The characteristic ¹³ C ssNMR chemical shifts of the Form 1 Compound A spectrum, expressed in ppm, are observed at 22.9 m .d., 146.2 ppm, 157.9 ppm, 161.9 ppm and 172.9 ppm±0.2 ppm The chemical shifts (± 0.2 ppm) in the ¹³ C ss NMR spectrum of Form 1 of Compound A shown in Figure 5 are shown in Table 4.

Table 4. 13C Form ¹ chemical shifts of Compound A and their intensity

¹³ C chemical shifts (ppm) Intensity ¹³ C chemical shifts (ppm) Intensity ¹³ C chemical shifts (ppm) Intensity 20.1 95 56.4 84 137.5 62 22.9 90 59.1 27 137.9 59 28.4 66 61.2 55 144.9 44 34.3 68 107.1 65 146.2 48 37.7 100 118.9 42 157.9 36 40.8 63 126.6 59 161.9 44 42.5 76 127.3 70 168.6 36 44.3 66 130.2 47 172.9 38 51.6 87 131.7 92 187.7 56 53.6 96 132.3 56

Form 2 of compound A is a trihydrate and is thermodynamically stable above the activity of water by about 0.2 at ambient temperature and 20% RH. Form 2 XRD of Compound A is the same as that shown in Figure 6. Characteristically, Compound A Form 2 peaks on XRD at the following 2-theta angles (2Θ±0.2°): 8.4, 9.0, 10 .5, 15.0 and 24.7. The location of the peaks and their intensity in the PRDH presented in figure 6 are shown in table 5.

Table 5. Location of peaks in PRDH form 2 of compound A and their relative intensity

Degrees 2Θ
± 0.2° Relative intensity(%) Degrees 2Θ
± 0.2° Relative intensity(%) Degrees 2Θ
± 0.2° Relative intensity(%) 5.0 eleven 16.1 ten 26.9 71 8.4 eighteen 16.7 59 28.2 52 9.0 12 17.4 74 29.0 fifteen 10.0 6 17.8 13 29.4 eighteen 10.5 62 18.6 thirty 29.9 13 12.1 9 18.9 45 31.4 fifteen 13.3 46 19.9 93 31.7 16 13.7 45 20.1 fifty 32.4 fourteen 13.9 46 21.2 46 33.6 5 14.6 37 21.5 21 34.5 7 15.0 80 24.7 100 37.0 12 15.4 fifteen 25.2 97

The Raman spectrum of Form 2 of Compound A is essentially the same as the Raman spectrum shown in Figure 7. The Raman spectrum of Form 2 of Compound A shows characteristic peak shifts, expressed in cm ⁻¹ , at 562, 692, 984, 1225, 1507, 1557 and 1610±2 cm ^-1 . The positions of the peaks (± 2 cm ^-1 ) of form 2 of compound A and their normalized intensity (W=weak, M=medium, S=strong) of the spectrum presented in figure 7 are shown in table 6.

Table 6. The location of the peaks in the Raman spectrum of form 2 of compound A and their normalized intensity

Peak position
(cm ^-1 ) Normalized Intensity Peak position
(cm ^-1 ) Normalized Intensity Peak position
(cm ^-1 ) Normalized Intensity 123 W 864 W 1344 W 179 W 884 W 1369 W 232 W 931 W 1387 W 284 W 984 W 1410 W 405 W 1019 W 1433 W 441 W 1048 W 1460 W 481 W 1077 W 1480 W 562 W 1097 W 1507 W 620 W 1109 W 1557 M 628 W 1118 W 1610 S 639 W 1140 W 1670 W 650 W 1194 W 2884 W 667 W 1225 W 2916 W 692 W 1246 W 2946 W 710 W 1261 W 2995 W 758 W 1277 W 3073 W 790 W 1305 W 3108 W 839 W 1321 W

The ¹³ C ss NMR spectrum of Form 2 of Compound A is essentially the same as the ¹³ C ss NMR spectrum shown in Figure 8. The characteristic chemical shifts of the ¹³ C ss NMR spectrum of Form 2 of Compound A, expressed in ppm, are observed at 19.2 m .d., 149.5 ppm, 155.6 ppm, 163.8 ppm and 188.3 ppm±0.2 ppm Chemical shifts (± 0.2 ppm) ¹³ C ssNMR spectrum of form 2 of compound A, presented in figure 8, are shown in table 7.

Table 7. ¹³ C chemical shifts and their intensity for form 2 of compound A

¹³ C chemical shifts (ppm) Intensity ¹³ C chemical shifts (ppm) Intensity ¹³ C chemical shifts (ppm) Intensity 19.2 60 63.1 44 139.1 33 25.7 87 107.0 40 149.5 33 32.0 40 108.7 35 155.6 thirty 38.0 92 125.1 56 163.8 36 38.5 94 128.0 44 169.5 26 44.2 41 130.0 70 174.0 29 53.2 100 132.3 33 188.3 39 55.5 53 135.9 37 59.4 76 137.4 35

Based on the description provided herein, one of ordinary skill in the art will appreciate that Form 1 and Form 2 of Compound A can be uniquely identified by several different peaks in the spectra or contours thereof in various combinations. The following are typical examples of characteristic peak combinations that can be used to separately identify Form 1 and Form 2 of Compound A, these combinations should not be construed as limiting other combinations of peak parameters described herein.

Data confirming the presence of three water molecules in form 2 of Compound A was obtained using a Bruker D8 Venture diffractometer at room temperature (see Figure 9). The structure is determined by internal phasing using SHELX software in a monoclinic space group (Monoclinic class space group) P2 ₁ /c (Version 5.1, Bruker AXS, 1997). The structure is then refined by full-matrix least squares. All atoms that are not hydrogen atoms are determined and refined using anisotropic displacement parameters.

Hydrogen atoms located on nitrogen and oxygen atoms are determined from the difference Fourier map and refined with limited distances. The remaining hydrogen atoms are placed in calculated positions, allowing them to be on their carrier atoms.

The resulting R-index is 7.2%. The Fourier finite difference does not reveal missing or incorrectly determined electron density.

Table 8 presents data related to form 2 of compound A:

Table 8

Empirical formula C ₂₈ H ₃₀ N ₄ O ₅ C ₄ H ₁₁ NO ₃ 3H ₂ O Mass according to the formula 677.74 Temperature Room temperature Crystal system monoclinic Wavelength 1.54178 Å space group P2 ₁ /c Unit Cell Parameters a=17.6927(9) Å
b=13.2753(7) Å
c=14.6480(8) Å α=90°
β=92.451(3)°
α=90° Volume 3437.3(3) Å3 Z four Density (calculated) 1.310 mg/ ^m3 F2 Compliance Criteria 1.053 Final R indices [I>2sigma(I)] R1=0.0723, wR2=0.1835 R indices (all data) R1=0.1244, wR2=0.2110

Crystalline 2-amino-2-(hydroxymethyl)propane-1,3-diol salt 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine ]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid. This crystalline salt is commonly referred to as the tris salt of compound A.

Crystalline Tris-salt of compound A, where the ratio -methoxypyridin-2-yl)benzoic acid and salt is 1:1.

A crystalline Tris salt of Compound A, wherein the crystalline salt is an anhydrous crystalline salt.

An anhydrous crystalline Tris salt of Compound A, wherein the XRD of said anhydrous crystalline salt includes peaks at 2Θ diffraction angles of 9.6°, 10.7° and 11.3°±0.2°.

An anhydrous crystalline Tris salt of Compound A, wherein the Raman spectrum of said anhydrous crystalline salt includes peak shifts at 1511 cm ^-1 , 1561 cm ^-1 and 1615 cm ^-1 ±2 cm ^-1 .

An anhydrous crystalline Tris salt of Compound A, wherein the ¹³ C ss NMR spectrum of said anhydrous crystalline salt includes chemical shifts at 22.9 ppm, 146.2 ppm. and 161.9 ppm±0.2 ppm

An anhydrous crystalline Tris salt of compound A, wherein the analytical parameters of said anhydrous crystalline salt are selected from the group consisting of a Raman spectrum including peak shifts at 1511 cm ^-1 and 1615 cm ^-1 ±2 cm ^-1 , and a ¹³ C ss NMR spectrum including at least one chemical shift at 22.9 ppm, 146.2 ppm or 161.9 ppm±0.2 ppm

An anhydrous crystalline Tris salt of Compound A, wherein said anhydrous crystalline salt is substantially pure.

A crystalline tris salt of Compound A, wherein the crystalline salt is a trihydrate crystalline salt.

A trihydrate crystalline Tris salt of Compound A, wherein the PRDH of said trihydrate crystalline salt includes peaks at 2Θ diffraction angles of 8.4°, 9.0°, and 10.5°±0.2°.

A trihydrate crystalline Tris salt of Compound A, wherein the Raman spectrum of said trihydrate crystalline salt includes peak shifts at 1507 cm ^-1 , 1557 cm ^-1 and 1610 cm ^-1 ±2 cm ^-1 .

A trihydrate crystalline Tris salt of Compound A, wherein the ¹³ C ss NMR spectrum of said trihydrate crystalline salt includes chemical shifts at 19.2, 149.5 and 163.8 ppm±0.2 ppm.

A trihydrate crystalline Tris salt of Compound A, wherein the analytical parameter of said trihydrate crystalline salt is selected from the group consisting of

PRDH, including peaks at 2Θ diffraction angles of 8.4° and 9.0°±0.2°,

a Raman spectrum including peak shifts at 1557 cm ^-1 and 1610 cm ^-1 ±2 cm ^-1 , and

¹³ C ss NMR spectrum including at least one chemical shift at 19.2 ppm, 149.5 ppm. or 163.8 ppm±0.2 ppm

Trihydrate crystalline tris salt of compound A, where the analytical parameter of the specified trihydrate crystalline salt is selected from the group consisting of PRDH, including peaks at 2Θ diffraction angles of 8.4° and 9.0° ± 0.2°, and a Raman spectrum, including at least one peak shift at 1507 cm ^-1 , 1557 cm ^-1 or 1610 cm ^-1 ±2 cm ^-1 .

A trihydrate crystalline tris salt of compound A, wherein the analytical parameter of said trihydrate crystalline salt is selected from the group consisting of PRDH comprising peaks at 2Θ diffraction angles of 8.4° and 9.0°±0.2°, and ¹³ C ss NMR spectrum, including at least one chemical shift at 19.2 ppm, 149.5 ppm or 163.8 ppm±0.2 ppm

PHARMACOLOGICAL DATA

The following protocols are, of course, subject to change by those skilled in the art.

A randomized, 8 parallel group, diluent control study was conducted in male Sprague-Dawley reds (Charles River (Boston, MA)) to determine blood and liver TG levels. For 96 rats (weighing ~200 g) standard conditions are used; the rats are housed in cages of two and kept on a reversed 12:12 day/night cycle (lights off at 8 am). Upon receipt, rats are randomized to receive standard rat chow or Western diet chow and dose groups and a 14-day run-in period is established prior to the study during which rats are fed standard rat chow or chow Western diet. Standard laboratory rodent food, 5053, was obtained from LabDiet (PMI, St. Louis, Missouri). Western diet food, D12079Bi, was obtained from Research Diets (New Brunswick, NJ).

For these studies, a diluent (carrier) containing 0.5% (w/v) methylcellulose (MC, Sigma Aldrich; 274429) in deionized water is prepared. Stock solutions of Compound A (prepared from the Tris salt) or Compound D are prepared from the respective compound to obtain a concentration at which 10 ml of the solution provides the desired dose in mg/kg, where the average weight of the rats used is approximately 200 g.

Beginning on study day 1, rats were given oral (10 ml/kg) diluent (control) (0.5% MC (% w/v) in deionized water), low or high doses of Compound A (1 mg/kg or 10 mg/kg QD, respectively), low or high doses of compound D (5 mg/kg or 30 mg/kg BID, respectively), or co-administered low doses (compound A at 1 mg/kg QD and compound D at 5 mg/kg kg BID) or high doses (Compound A at 10 mg/kg QD and Compound D at 30 mg/kg BID) of Compound A and Compound D.

Samples for plasma analysis in the "after eating" state. Blood for postprandial TG concentrations is collected 2 hours post-dose (2 hours in the dark cycle) from the lateral tail vein, transferred to BD microcontainer tubes coated with ethylenediaminetetraacetic acid disodium salt (K ₂ EDTA) (PN365974) and centrifuged at 4°C. The resulting plasma samples are then analyzed on a Siemens Chemistry XPT clinical analyzer (Malven, PA) using Siemen triglycerides_2 reagents (ref 10335892).

Samples for analysis of blood plasma in the "fasting" state. Blood for determination of fasting plasma TG concentrations is collected after a 4-hour fast, 2 hours post-dose (2 hours in the dark cycle) from the lateral tail vein, transferred to BD microcontainer tubes coated with ethylenediaminetetraacetic acid dipotassium (K ₂ EDTA) (PN365974) and centrifuged at 4°C. The resulting plasma samples are then analyzed on a Siemens Chemistry XPT clinical analyzer (Malven, PA) using Siemen triglycerides_2 reagents (ref 10335892).

On the last day of the study (Day 28), rats are sacrificed for tissue collection after 4 hours of fasting, 2 hours post-dose: two hours post-dose, blood is collected via the lateral tail vein for plasma component determination, and then the animals are sacrificed by CO ₂ aspiration . Blood is transferred to BD microcontainer tubes coated with ethylenediaminetetraacetic acid dipotassium (K ₂ EDTA) (PN365974), centrifuged at 4°C, plasma transferred to a 96-well microtiter plate and stored at -20°C. The liver is quickly removed, frozen in a Wollenberg clamp, pre-chilled in liquid N ₂ , separately wrapped in aluminum foil, and stored at -80°C.

Tissue comminution: Frozen liver is rapidly comminuted on an aluminum block cooled in liquid N ₂ , thus ensuring that the tissue remains frozen during comminution. The shredded tissues are transferred to 7 ml polypropylene conical tubes and stored at -80° C. until analysis.

Hepatic triglyceride extraction: Approximately 50 to 100 mg of minced tissue is added to a 2 ml Matrix D Lysis Tube (MP Bio) containing 800 μl of ice-cold (1:1) CHCl ₃ :MeOH. Samples are immediately extracted at 4°C using Qiagen Tissue Lyser II (Qiagen Cat No. 85300) for 4 minutes at 30 Hz. After that, the homogenate is transferred into glass test tubes (13×100 mm) and placed on ice. The lysis tubes are then washed with 800 μl of a mixture (1:1) CHCl ₃ :MeOH, shaken for 30 seconds and the extract is transferred into glass tubes (13×100 mm). In all glass tubes placed on ice, add 2.4 ml of 100% CHCl ₃ to bring the ratio of CHCl ₃ :MeOH to 4:2. After that, the samples are kept in a cooling chamber at -20°C. The next day, 1.75 ml of 1M KCl H ₂ O was added to give a CHCl ₃ :MeOH:H ₂ O ratio of 4:2:1.75. The samples are shaken for 30 seconds and centrifuged (1500 rpm x 15 min) at 4°C. After centrifugation, the organic phase is transferred to a fresh extraction tube (13×100 mm), dried at 37° C. under N ₂ and resuspended in 750 μl CHCl ₃ . Aminopropyl solid phase extraction (SPE) cartridges (Waters Cat #054560, 6 mL, 500 mg) are wetted and washed with 5 mL of hexane. After washing, 200 μl samples of the extract in CHCl ₃ were applied to the cartridge and removed by vacuum without drying the column. Neutral lipids are then eluted with 5 ml of 2:1 CHCl ₃ :isopropanol/50 μM butylated hydroxytoluene. The samples are then dried at 37° C. under N ₂ and resuspended in 1.75 ml of a 98:2 mixture of isooctane:isopropanol. Samples are filtered through a syringe filter (0.2 μM) before injection into a Cyanopropy HPLC column (particle size 3.5 μM - 4.6 x 150 mm, Agilent Zorbax Eclipse XBD-CN column). Method: 4 µl injection for 27 minutes assay using solvent A (1000:1:2, isooctane:isopropanol:acetic acid) and solvent B (50:50 isopropanol:methyl tert-butyl ether). At 0-3 minutes solvent composition: 100% solvent A. Minutes 3-8: solvent composition changes from 100% solvent A to 95% solvent A and 5% solvent B. Minutes 8-18: solvent composition changes to a ratio of 50:50 . Minutes 18-19: The solvent composition changes again to 100% Solvent A and this composition is maintained for 19-27 minutes.

Fractions of nuclei and membranes are obtained from a portion of the crushed liver samples, which are combined into one treatment group, by ultracentrifugation using standard methods. Samples of nuclear extract and membrane fractions are analyzed by Western blotting for SREBP1. Calnexin Western blots were used as a marker for the membrane fraction, actin as a marker for total sample loading, and histone 2B as a marker for the nuclear fraction. SREBP1 nuclear levels are quantified using relative units and normalized to histone 2B to control sample loss during nuclear fractionation and gel loading.

The other part of the crushed liver is processed and analyzed for the expression of lipogenic genes. Rat linear taqman probes for AKK1, FASN, SCD1, PCSK9 and SREBP-1c were evaluated using Actb as the qPCR housekeeping gene.

Administration of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide (compound D) or its pharmaceutical acceptable salt in combination with 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridine-2 -yl)benzoic acid (Compound A) or a pharmaceutically acceptable salt thereof resulted in a significant reduction in plasma (Figures 10, 11) and liver (Figure 18) TG levels relative to plasma and liver TG levels when Compound A was administered as monotherapy.

Feeding according to the Western diet resulted in a 2.2-fold increase in plasma TG levels in fasted rats compared to rats fed a standard diet (Figure 10). Oral low dose (1 mg/kg QD) or high dose (10 mg/kg QD) oral administration of Compound A alone (monotherapy) resulted in a 1.7-fold and 1.3-fold increase in plasma TG levels, respectively, according to compared to rats fed a Western diet fed with diluent. Conversely, oral administration of compound D alone (monotherapy) at low (5mg/kg BID) or high (30mg/kg BID) dose reduced postprandial plasma TG by 55% and 63%, respectively, relative to rats. who are fed a western diet and are fed a diluent. Co-administration of Compound A and Compound D results in complete blockade of the Compound A-mediated increase in plasma TG in the postprandial state. Oral co-administration of Compound A and Compound D at low or high doses of each resulted in a 37% and 64% reduction in postprandial plasma TG levels, respectively, compared to Western diet-fed rats administered with diluent.

Feeding according to a Western diet resulted in a 1.6-fold increase in fasting plasma TG compared to rats fed a standard diet (Figure 11). Oral administration at low and high doses of Compound A as monotherapy resulted in a 2.4-fold and 1.7-fold increase in fasting plasma TG levels, respectively, compared with Western diet-fed rats. enter the diluent. Conversely, oral administration of low and high doses of compound D as monotherapy resulted in a decrease in fasting plasma TG by 20% and 35%, respectively, compared to Western diet-fed rats fed diluent. Oral co-administration of Compound A and Compound D, at both low and high doses, completely suppresses the Compound A-mediated increase in fasting plasma TG observed with Compound A alone. Fasting" for both the low dose (109 mg/dL) and high dose (81 mg/dL) groups of Compound A and Compound D coadministration are similar to TG levels in rats fed a Western diet administered diluent (96 mg/dL).

The intranuclear localization of SREBP-1 is compared in samples of Western-fed rats treated with diluent, high-dose Compound A alone, Compound D high-dose alone, or Compound A and Compound D co-administered at high doses. (figure 12). In relation to diluent-fed western-fed rats, administration of Compound A results in increased nuclear localization of SREBP-1, indicating increased SREBP-1 activation. Administration of Compound D conversely results in decreased nuclear localization of SREBP-1 and activation of SREBP-1. Co-administration of Compound A and Compound D blocked the Compound A-mediated increase in nuclear localization of SREBP-1, resulting in a 50% reduction compared to Compound A alone.

Among diluent-fed rats, Western diet-fed rats show increased expression of lipogenic genes compared to standard chow-fed rats: AKK1 (Figure 13), FASN (Figure 14), SCD1 (Figure 15), and SREBP1 (FIG. 16) but not PCSK9 (FIG. 17), which is lower in Western-fed rats. Compared to diluent administration to animals fed a Western diet, Compound A administration tends to further increase the expression of ACC1, FASN (compound A high dose only), SCD, but not PCSK9 and SREBP1. Conversely, the introduction of compound D reduces the expression of all lipogenic genes. Co-administration of Compound A and Compound D results in expression levels that are comparable to or lower than those observed in diluent-fed rats fed a Western diet.

Compared to rats fed a standard diet and fed with diluent, rats fed a Western diet and fed diluent show an increase in hepatic triglyceride accumulation by about 2.7-fold (Figure 18). Oral administration of a low dose and a high dose of Compound A resulted in a 36% and 53% reduction in steatosis, respectively, compared to rats fed a Western diet fed with diluent. Similarly, oral administration of a low dose and a high dose of Compound D resulted in a 25% and 30% reduction in steatosis, respectively, compared to diluent-fed western-fed rats. Oral co-administration of a low or high dose of Compound A and Compound D reduced steatosis by 50% and 73%, respectively, compared to diluent-fed western-fed rats. Oral administration of a combination of Compound A and Compound D at low doses or high doses results in a greater reduction in steatosis than when Compound A or Compound D alone is administered at the same doses alone.

A 5-parallel, randomized, diluent-controlled, 5-parallel study was conducted in male Wistar-Han rats (Charles River (Boston, MA)) fed a choline-deficient and high fat diet - CDAHFD) (Research diets; A16092003) to identify differences in improvement in markers of inflammation and liver fibrosis when Compound A or Compound D is administered alone and when administered in combination. For 60 rats use standard conditions (~200 g); the rats are housed two per cage and kept on a reversed 12:12-hour day/night cycle (lights off at 8 am). Rats are fed on a low choline high fat (CDAHFD) diet starting 6 weeks prior to study entry. Rats randomized to 4 dosing groups (n=12/group) receive twice daily diluent, Compound A (5 mg/kg) as monotherapy, Compound D (30 mg/kg) as monotherapy, or a combination of Compound A ( 5 mg/kg) and Compound D (30 mg/kg) for 6 weeks. As a control group, animals (n=12) are used, receiving standard nutrition during the study, which are injected with a diluent twice a day. Blood samples are collected before starting the administration of the compounds and 3 and 6 weeks after the start of the administration of the compounds to evaluate circulating markers. Shear wave elastographic measurements (Aixplorer Ultimate imager, Supersoinc imagine) are performed at -3 weeks, 0 weeks (before the 1st dose), 3 weeks and 6 weeks to assess the development of inflammation and fibrosis over time. Histology is assessed after 6 weeks of drug administration, which corresponds to 12 weeks of feeding animals in accordance with CDAHFD. The results are presented as the mean of the animals per dose group.

After 12 weeks of feeding in accordance with CDAHFD, the animals are sacrificed by asphyxia in CO ₂ . Collect the right lateral, middle and left lateral lobes of the liver. Sections are taken from the left lateral, right middle and right lateral lobes of the liver of each animal, fixed in formalin and processed into paraffin blocks. One section of the left lateral lobe per animal is cryopreserved at optimal cut temperatures (OCT). The liver residue from each animal is frozen and rapidly ground on an aluminum block cooled in liquid N ₂ , thus ensuring that the tissue remains frozen during the milling. The shredded tissues are transferred and stored at -80° C. until analysis. A portion of a minced liver sample from each animal is processed and analyzed for fibrogenesis gene expression markers. Rat linear taqman probes for SMA and COL1A1 were evaluated using Actb as the qPCR housekeeping gene.

The following endpoints are assessed by a qualified histological evaluation by a certified veterinary pathologist and quantitative histomorphometry: activation of hepatic stellate cells and differentiation into myofibroblasts by immunohistochemistry αSMA (immunohistochemistry - IHC); collagen as a correlate of fibrosis using Picrosirius Red. Images are analyzed using Visiopharm software. Visiopharm applications with cut-off parameters are applied evenly to identify tissue sections and quantify targets on each IHC (DAB (3,3'-diaminobenzidine) positive) or histochemically stained slides as area percentage: stained area of interest/total tissue ROI - void area) × 100%. Nonparametric statistics are used to analyze the data obtained in this study. Group values are recorded as mean +/- standard error of the mean.

Compared to control animals fed the standard diet and given diluent, animals fed CDAHFD fed diluent show a significant increase in liver stiffness (determined using shearwave elastography (SWE) in pilopascals (kPa)) over the course of the study , indicating progressive inflammation and fibrosis of the liver (figure 19). Separate administration of Compound A or Compound D as monotherapy reduces liver stiffness, suggesting a reduction in liver inflammation and/or fibrosis. Co-administration of Compound A and Compound D resulted in a greater reduction in liver stiffness than either compound administered alone as monotherapy (FIG. 19).

Compared to control animals fed the standard diet and given diluent, animals fed CDAHFD and given diluent show a significant increase in liver alpha-smooth muscle actin (αSMA) staining indicative of fibroblast activation and fibrogenesis (Figure 20). Separate administration of Compound A or Compound D as monotherapy reduced αSMA staining by 41% and 23%, respectively, suggesting a reduction in hepatic myofibroblast activation and fibrogenesis. Co-administration of Compound A and Compound D resulted in a greater reduction in αSMA staining than administration of each compound alone when used as monotherapy, reducing staining by 72% (Figure 20).

Compared to control animals fed the standard diet and treated with diluent, animals fed CDAHFD treated with diluent show a significant increase in Picosrius red (PSR) staining indicative of collagen deposition and fibrosis (Figure 21). Separate administration of Compound A or Compound D as monotherapy reduced PSR staining by 26% and 20%, respectively, suggesting a reduction in collagen deposition and fibrosis. Co-administration of Compound A and Compound D resulted in a greater reduction in PSR staining than administering each compound alone as monotherapy, reducing staining by 56% (Figure 21).

Compared to control animals fed the standard diet and treated with diluent, animals fed CDAHFD and treated with diluent show a significant increase in staining of the ionized calcium binding adapter molecule 1 (Iba1), indicating hepatic macrophage activation (Figure 24). The introduction of compound A as monotherapy reduces Iba1 staining by 15%, suggesting a decrease in inflammatory liver tone. Although administration of D as monotherapy did not alter Iba1 staining, co-administration of A and D resulted in a greater reduction in Iba1 staining than administration of Compound A alone as monotherapy, reducing staining by 33% (Figure 24).

Compared to control animals fed the standard diet and fed diluent, animals fed CDAHFD fed diluent show a significant increase in gene expression of α-alpha smooth muscle actin (αSMA) (Figure 22) and A1A (COL1A1) (figure 23), indicating the activation of myofibroblasts and fibrogenesis. Separate administration of Compound A or Compound D as monotherapy reduces the gene expression of hepatic αSMA and COL1A1, suggesting a reduction in hepatic myofibroblast activation and fibrogenesis. Co-administration of Compound A and Compound D resulted in a greater reduction in hepatic αSMA (Figure 22) and COL1A1 (Figure 23) gene expression than when each compound was administered alone as monotherapy.

In the present description, references are made to various publications. The contents of these publications are incorporated herein by reference in their entirety for all purposes.

Those skilled in the art will appreciate that various modifications and variations may be made to the invention without departing from the scope or spirit of the present invention. Other embodiments will be apparent to those skilled in the art from a consideration of the description and embodiments of the invention disclosed herein. It is intended that the description and examples be taken as illustrative only of the invention, the true scope and spirit of which is set forth in the following claims.

Claims (36)

1. Pharmaceutical composition for the treatment of a disease or condition selected from fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver and non-alcoholic steatohepatitis with cirrhosis of the liver and hepatocellular carcinoma or metabolic disease, comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof in combination according to with at least 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl ) benzoic acid or a pharmaceutically acceptable salt thereof, each compound in a therapeutically effective amount, in admixture with a pharmaceutically acceptable excipient. 2. Pharmaceutical composition for the treatment of a disease or condition selected from fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver and non-alcoholic steatohepatitis with cirrhosis of the liver and hepatocellular carcinoma or metabolic disease, comprising (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof, 4- (4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or its a pharmaceutically acceptable salt; and a pharmaceutically acceptable excipient. 3. The composition of claim 2 further comprising at least one additional pharmaceutical agent selected from the group consisting of an anti-inflammatory agent, an antidiabetic agent, and a cholesterol/lipid modulating agent. 4. A method of treating a disease or condition selected from fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver, and non-alcoholic steatohepatitis with cirrhosis of the liver and with hepatocellular carcinoma or metabolic disease, and the specified method comprises administering to a patient in need of such treatment a therapeutically effective amount of ( S )-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine -5-carboxamide, or a pharmaceutically acceptable salt thereof, in combination with at least a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine ]-1'-carbonyl)-6-methoxypyridin-2-yl)-benzoic acid or a pharmaceutically acceptable salt thereof. 5. A method of treating a disease or condition selected from fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver, and non-alcoholic steatohepatitis with cirrhosis of the liver and hepatocellular carcinoma or metabolic disease, wherein said method comprises administering to a person in need of such treatment a therapeutically effective amount of ( S )-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine -5-carboxamide or a pharmaceutically acceptable salt thereof and 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)- 6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof. 6. The method of claim 5 wherein the disease or condition is fatty liver. 7. The method of claim 5, wherein the disease or condition is non-alcoholic fatty liver disease. 8. The method of claim 5 wherein the disease or condition is non-alcoholic steatohepatitis. 9. The method of claim 5, wherein the disease or condition is non-alcoholic steatohepatitis with liver fibrosis. 10. The method of claim 5, wherein the disease or condition is non-alcoholic steatohepatitis with cirrhosis. 11. The method of claim 10, wherein the disease or condition is non-alcoholic steatohepatitis with cirrhosis of the liver and with hepatocellular carcinoma. 12. The method of claim 5, which further comprises administering a therapeutically effective amount of at least one other pharmaceutical agent selected from the group consisting of an acetyl-CoA carboxylase (ACC) inhibitor, a diacylglycerol-O-acyltransferase 1 (DGAT-1) inhibitor ), monoacylglycerol-O-acyltransferase inhibitors, phosphodiesterase-10 (PDE-10) inhibitor, AMPK activator, sulfonylurea, meglitinide, α-amylase inhibitor, α-glucoside hydrolase inhibitor, α-glucosidase inhibitor, PPARγ agonist, PPAR α/γ agonist, biguanide, glucagon-like peptide (GLP-1) modulator, liraglutide, albiglutide, exenatide, albiglutide, lixisenatide, dulaglutide, semaglutide, protein tyrosine phosphatase-1B (PTP-1B) inhibitor, SIRT-1 activator, dipeptidyl peptidase IV (DPP-IV) inhibitor, stimulant secretion of insulin, fatty acid oxidation inhibitor, A2 antagonist, c-Jun N-terminal kinase (JNK) inhibitor, glucokinase activators (GKa), insulin, insulin-like agents a, glycogen phosphorylase inhibitor, VPAC2t receptor agonist, SGLT2 inhibitors, glucagon receptor modulators, GPR119 modulators, FGF21 derivatives and analogs, TGR5 receptor modulators, GPBAR1 receptor modulators, GPR40 agonists, GPR120 modulators, high affinity nicotinic acid receptor activators (HM74A), SGLT1 inhibitors , inhibitors or modulators of carnitine palmitoyltransferase enzymes, fructose-1,6-diphosphatase inhibitors, aldose reductase inhibitors, mineralocorticoid receptor inhibitors, TORC2 inhibitors, CCR2 and/or CCR5 inhibitors, PKCα inhibitors, PKCβ inhibitors, PKCγ inhibitors, fatty acid synthetase inhibitors, serine palmitoyl transferase inhibitors, GPR81, GPR39, GPR43, GPR41, GPR105, Kv1.3 modulators, retinol-binding protein-4, glucocorticoid receptor, somatostatin receptors, PDHK2 or PDHK4 inhibitors or modulators, MAP4K4 inhibitors, IL1beta modulators, HMG-CoA reductase inhibitors, squalene synthetase inhibitors, fi siblings, bile acid sequestrants, ACAT inhibitors, MTP inhibitors, lipoxygenase inhibitors, cholesterol absorption inhibitors, PCSK9 modulators, cholesterol ester transport protein inhibitors, and RXRalpha modulators. 13. A method of treating fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver, or non-alcoholic steatohepatitis with cirrhosis of the liver and hepatocellular carcinoma in humans, comprising the step of administering to a person in need of such treatment, therapeutically an effective amount of the first agent, the second agent, and optionally the third agent, where: i. the first agent comprises ( S )-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof ; ii. the second agent includes 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl ) benzoic acid or a pharmaceutically acceptable salt thereof; and iii. the third agent includes a pharmaceutical agent selected from the group consisting of an anti-inflammatory agent, an antidiabetic agent, an antifibrotic agent, an antisteatotic agent, a cholesterol/lipid modulating agent, and an antidiabetic agent. 14. The method of claim 5, wherein -carboxamide is a solid crystalline substance of the structure:

or a pharmaceutically acceptable salt thereof. 15. The method of claim 14, wherein the powder X-ray diffraction pattern of the crystalline solid comprises peaks at 2-theta (CuKα radiation, wavelength 1.54056 Å) 5.3 ± 0.2, 7.7 ± 0.2 and 15.4 ± 0.2. 16. The method of claim 14, wherein the X-ray powder diffraction pattern of the crystalline solid comprises peaks at 2-theta (CuKα radiation, wavelength 1.54056 Å) 6.5 ± 0.2, 9.3 ± 0.2 and 13.6 ± 0.2. 17. The method according to p. 5, in which 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)- 6-methoxypyridin-2-yl)benzoic acid is a solid crystalline substance with the structure:

or a pharmaceutically acceptable salt thereof. 18. The method of claim 17 wherein the crystalline solid is 2-amino-2-(hydroxymethyl)propane-1,3-diol salt 4-(4-(1-isopropyl-7-oxo-1,4, 6,7-tetrahydrospiro[indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid. 19. Pharmaceutical composition according to claim 2, in which 5-carboxamide is a crystalline solid with the structure:

or a pharmaceutically acceptable salt thereof. 20. The pharmaceutical composition according to claim 19, wherein the X-ray powder diffraction pattern of said crystalline solid includes peaks at 2-theta (CuKα radiation, wavelength 1.54056 Å) 5.3 ± 0.2, 7.7 ± 0, 2 and 15.4 ± 0.2. 21. A pharmaceutical composition according to claim 19, wherein the X-ray powder diffraction pattern of said crystalline solid includes peaks at 2-theta (CuKα radiation, wavelength 1.54056 Å) 6.5 ± 0.2, 9.3 ± 0, 2 and 13.6 ± 0.2. 22. Pharmaceutical composition according to claim 2, wherein 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl) -6-methoxypyridin-2-yl)benzoic acid is a solid crystalline substance with the structure:

or a pharmaceutically acceptable salt thereof. 23. The pharmaceutical composition of claim 22 wherein the crystalline solid is 2-amino-2-(hydroxymethyl)propane-1,3-diol salt 4-(4-(1-isopropyl-7-oxo-1,4 ,6,7-tetrahydrospiro[indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid. 24. The pharmaceutical composition of claim 22, wherein at least one additional pharmaceutical agent is selected from the group consisting of [(1R,5S,6R)-3-{2-[(2S)-2-methylazetidin-1-yl ]-6-(trifluoromethyl)pyrimidin-4-yl}-3-azabicyclo[3.1.0]hex-6-yl]acetic acid; 2-[(1R,3R,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1 ]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acid; or 2-[(4-{6-[(4-cyano-2-fluorobenzyl)oxy]pyridin-2-yl}piperidin-1-yl)methyl]-1-[(2S)-oxetan-2-ylmethyl] -1H-benzimidazole-6-carboxylic acid or a pharmaceutically acceptable salt thereof. 25. A method of treating a disease or condition selected from fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis of the liver and non-alcoholic steatohepatitis with cirrhosis of the liver and hepatocellular carcinoma or metabolic disease, and the specified method comprises administering to a person in need of such treatment a composition according to claim 2.

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